JP2005270109A - Selection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for the isolation, creation or directed evolution of a gene encoding a novel molecule capable of having a desired interaction with a substrate of interest. <P>SOLUTION: In a method for selecting a molecule catalyzing cleavage of peptides, wherein the method comprises steps of (a) expressing a selection molecule including different putative molecules in a population of host cells and a recognition sequence binding to a generated peptide which is formed by cleavage of a starting peptide through molecules evolved from the putative molecule, (b) subjecting the population of host cells to mutagenizing conditions and selection conditions to select a replicator or a host cell so that at least one replicator and/or cell in the population undergoes at least one replication cycle and the multiple putative molecules evolve into a molecule catalyzing the cleavage of peptide, and (c) identifying the host cells expressing the molecule catalyzing the cleavage of peptide by monitoring the increase of proliferation of cells and/or replicators. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention is a rational method for isolating, creating or directing the evolution of a gene that expresses a novel molecule that performs a desired interaction with a target substrate using genetic recombination techniques and selections. Widely involved.

  More particularly, the present invention relates to proteins, peptides, nucleic acids, oligonucleotides, lipids used as reactants, catalysts, enzyme cofactors, repressors, enhancers, hormones and binders in various substrates of industrial and therapeutic products. And methods for isolating, creating, or evolving novel molecules, including organic, inorganic, and biomolecules such as polysaccharides.

More particularly, the present invention relates to a host cell or virus having a gene that expresses a novel molecule of interest by subjecting a host cell and / or virus expressing: Regarding the method of selecting variants:
-A regulated growth factor for the host or virus that is functionally associated with the target substrate or analog thereof; and-the ability to interact with the target substrate or analog thereof to alter the activity of the growth factor. A large number of copies of one putative novel molecule or a variety of putative multiple novel molecules.

  The methods of the present invention can be used for molecules with a wide range of interesting properties, such as various industrial, research or therapeutic catalysts, proteases, binding peptides, enzyme cofactors, enhancers, repressors, hormones, etc. Can be used to reasonably create.

  In this application, several publications are referenced in parentheses with Arabic numerals. Full citations for these references are at the end of the specification and immediately before the claims. References describe in more detail the state of the art to which the present invention pertains and certain aspects of the present invention.

  In general, there are three ways to obtain molecules with novel properties. The first method, eg protein engineering, relies on a theoretical model that seeks to reveal the known properties of a general type of molecule and the conformation of the molecule most likely to have the desired properties. None of the models have been proven sufficiently general or accurate to design the appropriate molecule in a reproducible manner.

The second method is screening. Screening requires testing multiple combinations of molecules for a given property. The current state of screening technology and the vast number of different combinations limit the usefulness of this technology. For example, a peptide sequence consisting of 20 amino acids has 20 ²⁰ different combinations. Screening bacteria that produce peptides of considerable length in different combinations would require billions of petri dishes each containing roughly a thousand colonies. It is extremely inefficient to screen such a large population and find only a few that have the desired properties, if any. Screening techniques are inadequate for the practical implementation of such issues.

The third method employs natural selection in a specific, non-generalizable way. For example, if a single cell organism lacks an enzyme in a critical metabolic pathway, mutants can be used to attempt to select molecules that have the same function as the missing enzyme. However, this technique is limited by the reactions encoded in the genome of the organism and the reactions that may be captured within the cell. In addition, a new mutant is required for each different supplementary experiment.
Methods for selecting prior art organisms are known in the art. These methods include, for example, selecting organisms that express molecules essential for cell growth, such as culturing host cells with organic compounds that cannot be utilized by the parent strain in the absence of essential nutrients, or in the presence of toxic analogs. Incubating is included. Such techniques are primitive. Because growth in the absence of essential nutrients does not allow researchers to rationally design molecular selection procedures for any particular type of reaction and for any particular target region within the substrate. Absent. Selection pressure based on growth in the absence of certain essential nutrients does not impose any reasonably defined selection pressure by which a growth advantage or disadvantage is conferred, and thus various molecules with a wide range of functions It is crude in that a host that survives expression may be selected. This limits the usefulness of such methods as it reduces the host's ability to isolate or create molecules with a particular desired ability.

  For example, growth with organic compounds that cannot be utilized by the parent strain is limited. This is because the host is selected based solely on the ability to utilize the organic compound. The use of the organic compound can be achieved by any of a number of different reactions. There is no rational way to isolate, create, or direct the evolution of molecules capable of specific reactions with target regions within specific substrates.

Dube et al., Biochemistry , Vol. 28, No. 14, July 11,1989 disclose remodeling of the gene encoding β-lactamase by replacing DNA with a random nucleotide sequence at the active site. Oligonucleotide substitutions preserve certain codons critical to activity, but contain chemically synthesized random sequence base pairs that encode over a million amino acid substitutions. A population of E. coli was infected with plasmids with these random inserts and incubated in the presence of carbenicillin and related analogs. Seven new active site mutants were selected that render the E. coli host resistant to carbenicillin, each mutant containing a multi-nucleotide substitution encoding a different amino acid. Each mutant showed temperature-sensitive β-lactamase activity. Dube et al. Are thus limited to enhancing the already known functions of a class of enzymes.

  Methods for producing novel molecules and DNA and RNA sequences by recombinant techniques and selection are disclosed in Kauffman et al., U.K. Patent Application No. GB 2183661A, filed on June 17, 1985. The mutated gene is introduced into the host cell and the transformed host is propagated so that the mutated gene is cloned, thereby facilitating the production of the protein expressed by the gene. The altered host cells are screened and / or selected to identify strains of host cells that produce new proteins with the desired properties, and the identified strains are propagated to produce new molecules with the desired properties. The technique taught by Kauffman et al., Like Dube et al., Is limited to methods that alter the known function of a class of molecules.

Schatz et al., Cell , Vol. 53, pp. 107-115 (1988) discloses a method for identifying a fibroblast cell line capable of expressing a gene encoding an enzyme with known recombinase activity. Yes. This method is based on a somatic recombination method in which widely separated gene fragments are joined together to form a complete variable region. [This variable region is assembled in an ordered and highly regular manner from V (variable), J (participating) and in some cases D (variable) fragments. In order to stably impart the ability of V (D) J rearrangement to fiber buds, gene transfer is used.

  A recombinase gene, that is, a retrovirus-based DNA recombination substrate comprising a gene library that encodes a gene that expresses an enzyme that plays a role in V (D) J recombination. Was transfected into a host cell having a gene expressing. Initially, genes that expressed growth factors were not transcribed or translated. However, when recombinase activity was expressed by the interaction of the recombinase and the recombinase recognition sequence, transcription and translation of the growth factor was activated.

Bock et al., Nature , Vol. 355, pp. 564-567 (1992) report an attempt to select a DNA molecule having a novel function. Aptamers, which are probabilistically generated oligonucleotides capable of binding specific target molecules, were selected by a cell-free selection method. Single-stranded DNA can be screened for aptamers that bind human thrombin, a protein whose nucleic acid binding function is not known at all. These processes that actually constitute a cell-free screening method include screening and amplification of certain members of the subpopulation. Other members are discarded.

Curtiss' PCT application No. WO89 / 03427 discloses methods and techniques for expressing recombinant genes in host cells. Curtiss discloses genetically engineered cells that express the desired gene product because they are maintained in a genetically stable population. The genetically engineered cell has the following characteristics:
(1) deletion of a gene encoding an enzyme essential for cell wall growth, that is, it cannot catalyze in one step in the biosynthesis of essential cell wall structural components;
(2) the first recombinant gene encodes an enzyme that is a functional replacement of the enzyme essential for cell wall growth; and (3) the second recombinant gene is the first recombinant It encodes the desired polypeptide that is physically associated with the gene.
Loss of the first recombinant gene causes lysis when the cell is placed in an environment free of the product in which the gene is expressed, and lyses cells cultured in an environment free of the first recombinant gene. cause.

  Baum et al., Proc. Natl. Acad. Sci., (USA), Vol. 87, pp. 10023-10027 (1990) relates to a method for monitoring cleavage interactions by various proteases. A fusion construct is created by inserting a protease cleavage site, such as the decapeptide human immunodeficiency virus (HIV) protease recognition sequence, into a specific site of E. coli β-galactosidase. These construct genes that retain their enzymatic activity despite insertion of the cleavage site are subcloned into plasmids encoding wild type and mutant HIV proteases, respectively. The fusion construct was found to be cleaved by wild type HIV protease, but not by mutant HIV protease, in both in vivo and in vitro experiments.

  As a result of cleavage by HIV protease, the altered β-galactosidase is inactivated. The cleavage reaction is inhibited by pepstatin A, a known inhibitor of HIV protease. A similar construct was developed using the polioprotease cleavage site, which was cleaved by the polioprotease.

  Paoletti et al., US Pat. No. 4,769,330, discloses a method for altering the vaccinia genome, particularly by introducing foreign DNA into the vaccinia genome, in order to generate vaccinia mutants. Unaltered and genetically altered microorganisms involved in this method are disclosed as DNA sequences and intermediates, and cells and host animals are infected with vaccinia mutants and encoded by foreign DNA A method for amplifying a protein is also disclosed. This reference is representative of known recombinant techniques used to alter both viruses and host cell microorganisms.

  Murphy, US Pat. No. 5,080,898 relates to the use of recombinant DNA techniques in the production of toxin molecule analogs, and the use of the analog molecules to treat medical disorders. The toxin molecule can bind to any specific binding ligand, whether it is a peptide or not, at the same predetermined position for any toxin molecule.

  Anderson et al., US Pat. No. 4,403,035, discloses a method for delivering and introducing genetic information by packaging a hybrid DNA protein complex into a viral vector and then introducing this genetic information into a susceptible microorganism from the hybrid virus. doing. An organism having the function or ability desired to be introduced is selected, and its DNA is isolated / purified and cleaved to isolate foreign genes that control the desired function so that they can be introduced or cloned. These foreign DNAs are inserted into the viral DNA. The hybrid DNA protein thus obtained is combined with the resources of the viral capsid precursor structure, ie prohead, and the additional viral structural and packaging proteins necessary to assemble the infectious hybrid virus encapsidating the hybrid DNA. Introduce into cell-free in vitro medium.

  Virus capsid precursor structure, and additional virus structural proteins and packaging proteins infect sensitive microorganisms with the first virus variant that can produce virus capsid precursor structure but does not produce at least one packaging protein And by infecting compatible microorganisms with a second viral variant that can produce additional viral structural proteins and packaging proteins, but not the capsid precursor structure. The infected cells are then mixed and lysed to provide a viral component resource for in vitro packaging of the hybrid DNA protein.

  The hybrid virus is then infected with a microorganism compatible with the virus and the infected cells are programmed to continuously reproduce the desired function of the foreign gene and the gene itself as a nucleic acid.

  Dulbecco, US Pat. No. 4,593,002, discloses a method for incorporating a DNA fragment into a viral DNA gene. A DNA fragment encodes a protein with a particular medical or commercial use. A small fragment of the original protein is identified that exhibits the desired function, and a DNA fragment having a nucleotide base sequence that encodes the protein fragment is isolated / purified from a biological or chemical synthesis. The isolated / purified DNA fragment should be such that the inserted DNA fragment is expressed as a foreign part of the viral surface protein and that the function of the protein fragment and any viral protein functions important for viral replication are not impaired. , Inserted into the viral DNA genome.

  All of the prior art methods provide a rational method that employs a selection process for the isolation, creation, or creation by directed evolution of new molecules with specific functions for a selected target substrate. It is not a thing. Screening methods are inherently inefficient, wasteful and time consuming. The primitive selection methods disclosed by the prior art do not allow the creation of molecules with a high degree of specificity for a particular recognition sequence, for example as a binder or as a catalyst. Those methods only create a limited number of molecules with limited properties. Furthermore, none of the prior art references teaches a universal method in applicability. There is no method in the prior art for the isolation, creation or directed evolution of multiple genes, each expressing a different molecule with a reasonably designed activity against the substrate of interest.

  Therefore, it is a first object of the present invention to create a novel molecule capable of interacting with a target substrate.

  The related objects of the present invention also include relatively new molecules used as reactants, catalysts, enzyme cofactors, repressors, enhancers, hormones and substrate binders in prior art protein engineering, screening and selection methods. It is to create while avoiding the loss of time, effort, and failure.

Another object of the present invention is to utilize selection processes and genetic recombination techniques for the production of unknown molecules with new or improved known functions for a wide range of target substrates.

  Yet another related object of the present invention is to isolate a gene expressing a new molecule from an existing gene pool by adapting the interactive specificity of the new molecule to its specific target substrate.

  Furthermore, another related object of the present invention is to create novel molecules that interact with the target substrate by rational design of methods based on selection using specifically expressed molecules that incorporate the recognition sequence of the target substrate. That is.

  Furthermore, another related object of the present invention is to take advantage of the rapid replication of cellular and viral genomes in the selection of genes that express a novel molecule of interest.

  In addition, another object of the present invention is to control and manage evolutionary pressures on cell and viral systems to achieve previously unknown physical and / or chemical interactions with the substrate of interest. To create and evolve genes that express these novel molecules.

  The present invention broadly relates to rational methods for the isolation, creation or directed evolution of genes that encode novel molecules capable of the desired interaction with the target substrate. This method involves the selection of a host that encodes the novel molecule or a replicator in the host, the selection being effected by the desired interaction of the novel molecule with the selected molecule expressed by the host, or growth of the cell or replicator. It is based on. This method is presumably put into a population of host cells containing a cell growth factor and / or a replicator (eg, viral) growth factor and a target substrate or analog thereof functionally associated with the growth factor. A novel molecule that expresses multiple copies of a novel molecule or a variety of different putative novel molecules, imposes selection conditions on this host cell population and interacts with the substrate of interest or analog thereof to alter the activity of the growth factor By selecting a host or replicator that expresses.

The invention also includes altered host cells used in the invention, altered replicators, certain selection molecules used to practice the methods of the invention, genes produced by the methods of the invention and novel Molecules and systems and kits useful for practicing the invention.
Host selection The isolation, creation or directed evolution of a gene encoding a novel molecule capable of the desired interaction with the target substrate can be performed by the following steps:
-Expressing multiple copies of a putative novel molecule or various putative novel molecules in a population of host cells;
-Adding or expressing a growth factor, a target substrate or analog thereof functionally associated with the growth factor having a recognition sequence corresponding to the substrate, and optionally a growth factor regulatory moiety;
-Imposing selection conditions on the host cell population to select a host with a gene capable of expressing a new molecule that interacts with the recognition sequence and alters the activity of a growth factor.

  The order in which the putative new molecule is expressed, and the order relative to each other and the expression of the putative new molecule in the expression and / or addition of the growth factor, the substrate of interest or analog thereof, and the regulatory moiety, And for any of these processes, the timing of the selection process is a matter of choice. In certain embodiments, the host or replicator is expressed to express a growth factor, recognition sequence or regulatory moiety, or a selection molecule incorporating them, in order to develop a desired host strain or replicator strain for later selection. It may be advantageous to impose selection conditions on the host or replicator population before changing the data rate.

The method of the invention can be carried out as follows:
A homogenous population of genes capable of expressing multiple copies of a putative novel molecule, or a heterogeneous population of genes capable of expressing a variety of different putative novel molecules or molecules with evolutionary potential, and cell growth factors and Introducing into a population of host cells whose genome has been artificially altered to express a recognition sequence functionally associated with the growth factor corresponding to the substrate of interest;
A host with a gene capable of expressing a new molecule that imposes selection conditions (eg, cultivates or incubates a host cell population under selection conditions) and interacts with a recognition sequence to alter the activity of a growth factor. Selected;
-Isolation / purification of the gene of interest from the selected cell population.
This target gene can then be used to express additional quantities of new molecules.

Growth factors and recognition sequences may be present as separate molecules or molecular groups, or may be present together in molecules that incorporate both. Host cells, such as E. coli, may be altered by externally adding growth factors and / or recognition sequences, or growth factors and / or recognition sequences may be expressed in the host cells. Selection of a host with a gene or variant thereof that can express a novel molecule that interacts with the recognition sequence and thereby affects the activity of the growth factor by imposing selection conditions on the host cell population Is possible.
Replicator selection method The isolation, creation or directed evolution of a gene encoding a novel molecule capable of a desired interaction with a target substrate can be carried out as follows. That is, a large number of copies of a putative novel molecule encoded by a replicator (eg, a virus) or a variety of different putative novel molecules are combined with a growth factor for the replicator and a recognition factor corresponding to the target substrate. Expressed in a population of host cells that contain or express a growth factor regulatory moiety that is functionally related to the substrate of interest, or an optional growth factor, and subject the selection A replicator, such as a virus, that can express a new molecule that interacts with the sequence and alters the activity of the growth factor is selected.

  These methods, for example, replicate a replicator, such as a virus, to artificially alter the genome to express a growth factor for the virus and a recognition sequence functionally associated with the growth factor corresponding to the substrate of interest. A virus capable of introducing a host cell population, expressing the novel molecule that interacts with a recognition sequence and alters the activity of a growth factor by culturing or incubating the host cell population, and selecting a gene of interest Can be performed by isolation / purification. Similar to the host selection method, in this method the order of expression and / or addition of several components in the above process and the order of expression and / or addition relative to the time when selection conditions are imposed is a matter of choice. It is.

In such a method, a homologous virus population that expresses multiple copies of a putative novel molecule, or a heterologous virus population that contains various mutant genes, each of which can express a different putative novel molecule, Are introduced into a modified host cell population containing growth factors and the above-described recognition sequences that have weakened functions required for growth and / or replication. Viruses that express new molecules that interact with the recognition sequence and thereby enhance the activity of growth factors will replicate in the host. Viruses that express new molecules that do not have the desired interaction will not replicate. The host cell can then be incubated or cultured under selective conditions to select a viral population that expresses the novel molecule of interest.

  In desirable methods, the genome of the host cell is artificially altered to express a molecule or molecules that contain a recognition sequence that is functionally associated with the growth factor and the substrate of interest. The host cell population is then replicated with a large number of copies of a molecule capable of interacting with a selection molecule expressed by the host cell's recombinant genome, or a genome capable of expressing such a variety of different molecules (eg, a virus). ). These novel molecules that interact with the recognition sequence and alter the function of the growth factor confer selective growth advantage to the virus expressing this selected novel molecule. The host cell population can then be cultured or incubated to obtain an amplified population of the desired virus.

  As in the case of host selection, the host cell genome is artificially altered to express growth factors and recognition sequences as separate molecules or as a physical or chemical association or combination thereof. The recognition sequence represents the substrate of interest and is functionally related to the replicator growth factor. Desirably, the genome of the host cell may be altered by genetic recombination techniques to express a selection molecule, ie, a fusion or deletion protein that contains both growth factors and recognition sequences. Growth factors and recognition sequences may be accompanied by selective moieties that modulate the activity of the growth factor. The selection portion may be an independent molecule or may itself be part of a selection molecule (eg, a fusion protein or deletion protein) that contains growth factors and recognition sequences.

  New molecules acquired may act through a cascade of events. That is, the molecule may interact with the recognition sequence to produce the desired effect, or the interaction initiates a cascade of events at any of the intermediate stages, which ultimately leads to growth factor activity. May affect. Each molecule in the cascade can be a natural or engineered substrate in the host cell, or an externally supplied substrate, or itself a novel molecule.

  The host selection method and replicator selection method of the present invention can be used to create a wide range of novel molecules, such as novel proteases capable of interacting with a protease recognition sequence in a desired manner. Molecules other than protease can also be produced. For example, an enzyme capable of site-specific glycosylation (or phosphorylation, etc.) in the vicinity of cellular proteins important for growth, particularly sensitive to glycosylation (or phosphorylation, etc.) and allowing the insertion of a recognition sequence. Can be created.

  Universal selection methods link the formation of virtually any compound to cell growth. For example, in the reaction A + B → C (catalyzed by X), even if C does not have any direct or indirect influence on cell growth, the production of C is linked to cell growth, Those that can catalyze the reaction (whatever the reaction type) from a group of compounds, can act as a substrate, or can act as the product C itself, that is, can act in some way to contribute to the production of C. You can choose.

The present invention provides significant advantages over the prior art. The present invention provides an inherent efficiency improvement over screening and places the burden on the experimental system rather than the experimenter. In selection, environmental conditions determine which members of the population can grow. By appropriately selecting the selection procedure and conditions, it is possible to obtain clones having desired characteristics from a large population. The selection procedure of the present invention has the advantage that each can be used to obtain a vast number of novel molecules that are highly specific for interaction with a given recognition sequence. In contrast, the primitive selection method is crude and empirical.
DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS Some terms and expressions used herein are defined as follows:
The term “isolated” means bringing a naturally occurring gene from an existing gene pool.

  The term “creation” means producing a gene that encodes a new molecule that does not exist in nature.

  The expressions “directed evolution” and “creation by directed evolution” suddenly make a gene encoding a novel molecule that does not exist in nature under rationally designed selection conditions and pressure. It means to produce by mutating.

  The expression “new molecule of new function” has a structure and / or sequence and / or physical / chemical properties that have not been known so far, and a reaction type that has not been known or realized before. Includes all molecules that have specificity (eg, phosphorylation, proteolysis, binding, etc.) and / or specificity.

  The expression “functionally propagated novel molecule” is a molecule having a structure and / or sequence and / or physical / chemical property that has not been known so far, and its function, ie phosphorylation, proteolysis, binding, etc. Combinations of these reaction types are known or realized, but include molecules that differ in the degree of known function.

  The term “novel molecule” refers to any molecule having a previously unknown structure and / or sequence and / or physical / chemical properties, or a novel molecule with a new function, or a functionally enhanced novel Including molecules, including organic, inorganic and biomolecules such as nucleic acids, proteins, oligonucleotides, saccharides, lipids, peptides, and substituted or modified versions thereof.

  The expression “putative novel molecule”, “putatively functionally propagated novel molecule” or “putative novel molecule with a new function”, whether known or not, is known to those skilled in the art. Any molecule that is considered a candidate for isolation, creation, or directed evolution by methods.

  The term “subject of interest” includes any naturally occurring or synthetic molecule on which a chemical and / or physical interaction is desired to occur, whether known or not. The target substrate may consist of organic or inorganic molecules, such as proteins, oligonucleotides, lipids and polysaccharides, or biomolecules. The target substrate is a compound that is a known substrate of, for example, enzymatic reactions, peptides, polypeptides, and various proteins, which is reacted, cleaved, joined, modified or substituted, or bound by a new molecule including.

  The term “analog of a target substrate” means a molecule or a part thereof that contains a recognition sequence that makes the molecule a functional analog of the target substrate.

  The term “gene” is used in its broadest and generally understood sense and includes any expressible nucleic acid, such as an oligonucleotide (DNA, RNA, etc.), and further a combination of genes or Includes set.

  The term “genome” refers to all one set of genes that can be expressed, including chromosomal genes, plasmids, transposons and viral DNA.

  The term “expression” has a generally understood scientific meaning and includes oligonucleotide replication, transcription, translation and reverse transcription.

  The expression “expression by the host” means expression by any part of the host genome.

  The expressions “interaction” and “capable of interaction” broadly encompass any intermolecular and / or intramolecular action including chemical reactions, catalysis or physical bonds. Novel molecules created by the methods of the present invention may be capable of reacting with a recognition sequence in any reaction including, for example, isomerization, addition, substitution, synthesis and decay, and the like. Other interesting “interactions” include (de) phosphorylation, (de) glycosylation, (de) hydroxylation, (de) adenylation, (de) acylation, (de) acetylation and stereoisomerization Is included. Such a reaction may be limited to the interaction between the new molecule and the recognition sequence, or from each new molecule and / or recognition sequence, and / or from the host cell or medium, respectively. May include addition or deletion of molecules. The interaction also includes catalysis, where the new molecule catalyzes the recognition sequence, specifically resulting in proteolysis, esterification, hydrolysis, stereoisomerization, or any of the above reactions . The term interaction also encompasses allosteric effects induced by binding interactions or binding, such as found between antibodies and antigens.

  The term “host cell” includes living organisms that are unicellular, multicellular, prokaryotic or eukaryotic, such as yeast, COS cells, CHO cells or hybridoma cells.

  The term “growth factor” as used herein is a molecule that confers a growth advantage or disadvantage to a host cell or replicator. Typical growth factors include nutrients, enzymes required for nutrient metabolism, binding and structural proteins, and proteins involved in replication, metabolism, formation and maintenance of essential structural components, or cellular and subcellular growth.

The term “recognition sequence” is used in its most general chemical sense and can be any chemical atom, bond, molecule, subgroup, any combination thereof, or any physical Or it means an electrical state or arrangement. An example is an amino acid sequence. The recognition sequence can be an independent molecule or it can be the submolecular portion of a complex molecule that includes growth factors and / or selected moieties. It is essential that the recognition sequence has some functional interaction either directly or indirectly with the growth factor and / or selected moiety. For example, when a desired interaction between a new molecule and a recognition sequence occurs, the function of the growth factor is affected and impacts (positive or negative) on the growth of the cell or replicator. is there. The function and / or structure of the recognition sequence and / or selection moiety may be linked, and the function and / or structure of the recognition sequence and / or growth factor may also be linked.
As described further below, fusion or deletion proteins are particularly useful in the selection methods of the present invention and represent embodiments in which all three functions are combined in a single molecule.

  The modification of the recognition sequence allows the selection of a large number of new molecules. For example, a large protein that is a target substrate may be able to withstand a series of amino acid insertions or substitutions without losing its function (29). In such proteins, it is possible to insert different potential proteolytic recognition sequences in order to obtain a large number of proteases with the desired specificity.

  In addition, it is essential that the recognition sequence corresponds to the target substrate. For example, the recognition sequence can be an amino acid sequence from a protein for which no proteolytic enzyme is known or for which there is not enough proteolytic enzyme. Incorporation of this amino acid sequence or analog thereof as a recognition sequence based on the method of the present invention creates a novel molecule with specificity and / or high turnover for proteolysis of the amino acid sequence.

  The term “selection moiety” or “regulatory moiety” refers to any molecule that physically or chemically associates with a growth factor to directly or indirectly increase or decrease the function of the growth factor. The selection moiety may be a large protein that inactivates or impairs the function of a cell or virus required for growth by, for example, steric hindrance and conformational change.

  The term “selection molecule” or “universal selection molecule” refers to a molecule that incorporates growth factors, recognition sequences, and, optionally, regulatory moieties.

  The term “artificial selection molecule” refers to a devised selection molecule having a recognition sequence that is not unique to natural or synthetic growth factors.

  The term “replicator” refers to the subcellular presence capable of replication. For example, plasmids having a recognition sequence for replicase, mycoplasma, etc., viruses, bacteriophages, self-replicating oligonucleotides such as RNA molecules, and the like. Replicator expression refers to expression performed by a replicator via a host and / or replicator component.

  The terms “selection”, “selection condition” and “selection pressure” are used in terms of temperature, light, pH tower, various special environmental conditions in the absence of essential nutrients for compounds not available to the parent company. In the presence of toxins, or in the presence of mixed cultures, this refers to the well-known as well as new procedures for growing a host cell population as if only some but not all of its members survive and replicate.

  “Nucleotides” are five bases, adenine, cytosine, guanine, thymine, and uracil with one sugar, deoxyribose or ribose, and one phosphate.

  An “oligonucleotide” is a sequence formed by at least two nucleotides, and a “polynucleotide” is a long oligonucleotide, RNA or DNA, which may or may not contain modified bases. In the art, the term oligonucleotide is generally used to represent a relatively short nucleic acid strand, while the term polynucleotide is generally used to represent a relatively long nucleic acid strand comprising a DNA or RNA chromosome or fragment thereof. However, there is no limitation on the use of these terms here, and there is no description of the length unless otherwise specified.

  The term “nucleic acid” refers to a polynucleotide of any length, including DNA or RNA genomes or fragments thereof, and may or may not include modified bases as described above.

  The term “isolation / purification” refers to an isolation, purification or extraction technique, and in the same way that these terms are commonly used, a gene or molecule is cell and / or replicator and / or A method of recovering from a culture medium.

The term “mutagenesis” refers to the technique of creating a homogenous population of genes, eg, by irradiation, chemical treatment, low fidelity replication, and the like.
DESCRIPTION OF THE FIGURES FIGS . 1A and 1B illustrate an embodiment of the host selection method of the present invention for the creation of a gene encoding a novel molecule or the directed evolution of the gene.

  Figures 2A, 2B and 2C illustrate embodiments of viral replicators of the present invention for the creation of genes encoding new molecules or the directed evolution of the genes.

  Figures 3A and 3B illustrate further embodiments of the invention for the creation of a novel hydroxylase based on binding interactions or the directed evolution of the hydroxylase.

  4A and 4B illustrate a cell-free embodiment of the present invention.

  1A and 1B, reference numeral 10 represents a host cell (E. coli) having chromosome 12. The host cells are engineered as described separately to produce deletion mutants 14 that lack the ability to express certain essential growth factors. E. coli deletion mutant 14 is further engineered to encode a selection molecule that incorporates the above essential growth factors and recognition sequences for weakly regulated host cells, as shown in reference number 16.

  Reference number 18 represents a plasmid, which is engineered to encode the T7 bacteriophage origin of replication and the low fidelity T7 replication mechanism as shown in reference number 20. Plasmid 20 is further engineered to encode a heterogeneous population of genes that express a putative new molecule. The plasmid thus engineered is designated by reference numeral 22.

  Plasmid 22 is introduced into transformed defective mutant host 16, which is cultured in a nutrient-rich non-restricted medium in an appropriate environment as shown at reference numeral 24. A plasmid that expresses a new molecule that performs the desired functional interaction with a selection molecule expressed by the deletion mutant host 16 is designated by reference numeral 26.

  When cultured in non-restricted medium rich in nutrients by incubator 24, a population of host cells 16 containing plasmid 22 and plasmid 26 grows. The population of transformed host cells is indicated by reference number 30.

  The cultured host cell population is then placed in a chemostat and selected in nutrient-restricted media. This confers a growth advantage on the transformed host cell 16 containing the plasmid 26 which encodes a novel molecule that interacts with the selection molecule and has the desired function of enhancing growth factors. The selected host cell population is indicated by reference numeral 32. As can be seen from the figure, the cells containing the plasmid encoding the novel molecule having the desired function described above had a selective growth advantage.

  Plasmid 26 is then isolated / purified from the selected cell population 32. The gene for the new molecule is cloned, sequenced and functionally characterized.

  2A, 2B and 2C, reference number 50 represents a plasmid containing a functional T7 bacteriophage gene. Plasmid 50 is introduced into an E. coli host cell 52 having chromosome 54. The transformed host is indicated by reference numeral 56.

  Wild type T7 bacteriophage is indicated by reference numeral 58. A population 60 of deletion mutant bacteriophages that do not encode growth factors essential for replicator growth or replication are engineered to encode putative multiple novel molecules.

  A heterogeneous population of T7 deletion mutants is introduced into transformed host cells 56 that complement the function of the deletion mutant, and the populations of these deletion mutants are incubated. A population of deletion mutants is indicated by reference numeral 62. One of the T7 mutants in the population, having a gene that expresses a novel molecule capable of the desired interaction, is indicated by reference numeral 64.

  Reference number 66 represents a plasmid having a selection molecule containing a growth factor deleted from phages 60 and 64, a recognition sequence, and a gene expressing a regulatory moiety. This plasmid 66 is introduced into an E. coli host cell 68 having chromosome 70 to form a second population of transformed E. coli host cells indicated by reference numeral 72.

  The T7 deletion mutant population grown in the first population of transformed E. coli host cells that compensates for the deletion is then infected with a second population of cells expressing the selected molecule. The infection stage is indicated by reference numeral 74 and the incubation of the infected second population of host cells is indicated by reference numeral 76. Viral replication occurs only in host cells in which a novel molecule with the desired function is expressed. This process can be carried out by batch operation, or, as shown, the mutant T7 bacteriophage can be continuously added until lysis is observed in the second population of transformed host cells. Lysis in the second population of transformed host cells incubated is indicated by reference numeral 78. As can be seen, the population of T7 deletion mutants 64 encoding a novel molecule with the desired function is substantially amplified as indicated by reference numeral 80.

  The viral population expressing the desired new molecule expands and infects other cells as a result of lysis. T7 is not newly added to the medium. This expansion and infection to other cells is indicated by reference numeral 82. Multiple infections further increase the viral particles with the desired novel molecule, along with viral particles that do not. This is indicated by reference numeral 84.

  A selected, amplified, viral population encoding a new molecule with the desired function (reference number 86) is isolated / purified from cultured host cells and then cultured on low dilution cells expressing the selected molecule. To do. This isolates individual virus clones with genes encoding novel molecules with the desired function. This isolation / purification is shown at reference number 88.

  The method shown in FIGS. 3A and 3B is for the creation or directed evolution of a hydroxylase that is site-specific for the substrate of general formula R, ie, a hydroxylase that can convert R to R—OH. It is a method. Reference numeral 110 represents a host cell (E. coli) having a cell wall 112, a periplasmic space 114, a cytoplasm 116 and a bacterial chromosome 118. The cell 110 contains a substrate R and an antibody specific for the compound R-OH indicated by reference numeral 120 in the periplasmic space, to which a regulated growth factor 122 is bound. Also present in the periplasmic space 114 is the binding conformation specific protease 124 of the antibody 122.

  A population of host cells 110 is transformed with a plasmid that replicates with a low fidelity replication mechanism that encodes a heterogeneous population of hydroxylases. The stage of infection is generally indicated by reference numeral 126. The infected host cell indicated by reference number 128 contains a number of plasmids 130 in the cytoplasm 116. These plasmids express a heterogeneous population of hydroxylases in the periplasmic space 114. A hydroxylase having the desired site-specific hydroxylase activity for substrate R is indicated by reference numeral 132, and an undesired hydroxylase is indicated by reference numeral 134.

  The population of transformed host cells is then incubated under selection conditions to select the desired hydroxylase or to direct its evolution. This step is generally indicated by reference numeral 136. A selected population of transformed host cells contains the hydroxylated substrate R, ie the compound R-OH shown in reference number 138. Subsequently, as shown at reference numeral 140, antibody 120 binds to this compound R-OH. Protease 124, specific for the binding conformation of antibody 140, enhances growth by cleaving regulated growth factor 122 from antibody 140 and conferring a growth advantage on cleaved antibody 142 and transformed host cell 128. Leave factor 144.

Next, a selected population of transformed host cells 128 is incubated to isolate / purify the DNA. The DNA encoding the desired hydroxylase is cloned and the desired hydroxylase is expressed and characterized.
Further described below with reference to several particular features of the present invention a preferred embodiment of the Detailed Description of the Invention.
I. Selection molecules and their component partial selection molecules are used to guide the selection pressure to obtain the desired gene. Selection molecules include growth factors and recognition sequences, and optionally regulatory moieties. Some can be used to select multiple different new genes by utilizing recognition sequences in a cassette-like form. Once a reasonable configuration of the desired component is established, steps are taken to prevent mutation of the selected molecule. This provides a constant target for the new group of molecules and helps guide the selection or evolutionary process.
A. Growth factors Growth factors are all factors that can give a cell or replicator a growth advantage or disadvantage. Growth factors are carbon sources, nitrogen sources, energy sources, phosphate sources, nutrients such as inorganic ions, nucleic acids, amino acids, etc .; antibiotics, toxins such as inhibitors of enzymes essential for replication, surfactants, etc .; cells Or an enzyme that is essential for replicator growth or that gives an advantage or disadvantage to cell growth, such as polymerases, ligases, topoisomerases, enzymes that catalyze reactions in protein biosynthesis; Molecules that are rather structural or based on the binding ability of the molecule, such as actin, lipids, nucleosomes, receptors, hormones, cyclic AMP, etc .; and water, inorganic ions, NADPH, coenzyme A, etc. Contain any coenzymes or cofactors.
B. Recognition sequences A recognition sequence is a molecule or part of a molecule that interacts with a new molecule. As such, the recognition sequence can take a variety of different structures, such as amino acid or nucleic acid sequences. This sequence may represent a single sequence or a plurality of related sequence types. The recognition sequence may be a special conformation or type of conformation of various molecules, for example a specific three-dimensional structure such as a protein, an inorganic molecule, a lipid, an oligosaccharide, etc. Furthermore, the recognition sequence may be an analog of the foregoing.

Further, depending on the selection system, the potential recognition sequences may be limited to very specific regions, conformations, sequences, etc., or may be a broad set of potential recognition sequences. For example, use a specific multiple overlapping sequence common to multiple selection molecules so that it and the desired new molecule interact to modulate growth, and the true recognition sequence is all of those selection molecules May be limited to a specific sequence common to the two. On the other hand, by using a single selection molecule or multiple selection molecules with a large region common to all, potential recognition sequences can be converted into a large region common to one selection molecule or multiple selection molecules. Various regions, conformations, sequences, etc. may be used. Thus, a recognition sequence may be highly specific for a region, conformation, sequence, etc., or more broadly still specific for a set of defined regions, conformations, sequences, etc. Also good.
C. Regulatory moiety The core of the present invention is the concept of modulation of growth factor activity. There are many ways to modulate biological activity, and nature has provided many precedents. Modulation of activity can be done through mechanisms ranging from complex and complex, such as allosterically induced quaternary changes, to simple presence / absence systems, such as expression / degradation systems. Indeed, the suppression / activation of the expression of many biomolecules is itself mediated by molecules whose activity can be modulated through various mechanisms.

  A table of chemical modifications to bacterial proteins is given on page 73 of (2). As shown in the table, some modifications are incorporated into the appropriate assembly and others are not, but in any case such modifications can cause a modulation of function.

  In some cases, modulation of functional utility can be mediated solely through proper / inappropriate localization of the molecule. Molecules can function only when they are directed to a particular location to provide a growth advantage or disadvantage. For example, starch is a macromolecule that is typically not absorbed by bacteria, and therefore it is necessary to secrete the enzymes necessary for its degradation, such as amylase, so that it can be converted to an available energy form. Thus, when bacteria are grown in starch-limited media, the production and maintenance of amylase in the bacteria down-modulates its functional utility. It is only when amylase is secreted that amylase can give the bacteria a growth advantage. The natural enzymatic ability of amylase can be the same whether it is inside or outside the bacterium, but when it is targeted inside the cell, it can be targeted outside the cell. In comparison, its functional utility is dramatically down-modulated.

  Protein localization targeting performed through cleavage of the signal peptide is one method that uses modulation of functional utility through molecular targeting within the scope of the present invention. In this case, a choice for specific endoprotease catalytic activity is chosen.

  The functional utility of an enzyme can also be modulated by changing its ability to catalyze the reaction. Such modulation can be done by differential localization (ie, acceptable local environment versus unacceptable local environment), but this need not be the mechanism described above. Specific examples of modulated molecules include zymogens, multi-subunit functional complex formation / dissociation, RNA virus polyprotein chains, allosteric interactions, general steric hindrance (covalent and non-covalent) ), And various chemical modifications such as phosphorylation, methylation, acetylation, adenylation and uridenylation ((2), p. 73, 315).

Zymogens are examples of natural protein fusions that cause modulation of enzyme activity. Zymogens are one type of protein that is converted to its active state via limited proteolysis ((3), p. 54). Nature has developed mechanisms to down-modulate the activity of certain enzymes, such as trypsin, by expressing these enzymes with an additional “leader” peptide sequence at their amino terminus. By having an extra peptide sequence, the enzyme is in an inactive zymogen state. Cleavage of this sequence converts the zymogen to its enzymatically active state. The overall reaction rate of zymogen is “about 10 ⁵ -10 ⁶ times lower than that of the corresponding enzyme” ((3), page 54).

  Thus, it is possible to down-modulate the function of certain enzymes simply by adding a peptide sequence to one of the ends of the enzyme. For example, this property can be used to select endoproteases having the desired properties within the scope of the present invention.

  Formation or dissociation of multi-subunit enzymes is another way through which modulation can occur. Various mechanisms may be involved in modulating activity in the formation or dissociation of multi-subunit enzymes. Two mechanisms can be illustrated.

  Tryptophan synthetase consists of two different subunits, α and β, in the α-β-α-β tetramer. This tetramer can dissociate into two α and β-β subunits, each of which exhibits enzymatic activity, although independent subunits are much less efficient than tetramer holoenzymes. One reason for the increased efficiency of holoenzymes is thought to be the formation of a tunnel between the α and β active sites (4). By determining the three-dimensional crystal structure of the enzyme, this tunnel sends the intermediate product of the α-catalyzed reaction directly to the β-subunit active site, thereby preventing loss to the solvent and thereby increasing efficiency. It became clear that

  For aspartate transcarbamoylase, modulation of activity in the formation of holoenzymes occurs through different mechanisms. In aspartate transcarbamoylase holoenzyme, the active site is formed on the opposing surface of the catalytic subunit. In both aspartate transcarbamoylase and tryptophan synthetase, appropriate specific interactions of different subunits are critical for the efficient activity of holoenzymes. Thus, sterically hindering the appropriate specific subunit interaction will down-modulate the catalytic activity. Such complexes can be used to select a variety of molecules within the scope of the present invention.

  Other examples of mechanisms through which functional modulation can occur include RNA viral polyproteins, allosteric effects, and general covalent and non-covalent steric hindrance. The HIV virus is a well-studied example of an RNA virus that expresses a non-functional polyprotein construct. In the HIV virus, “the gag, pol and env polyproteins are processed to produce the viral structural proteins p17, p24 and p15—reverse transcriptase and integrase, respectively—and the two envelope proteins gp41 and gp120” (see FIG. Five). Proper cleavage of the polyprotein is essential for viral replication, and virions with inactive mutant HIV protease are non-infectious (5). This is another example of a protein fusion that down-modulates its activity. Thus, it is possible to construct recombinant viruses that require sequence-dependent endoproteases for proper replication.

  Certain enzyme inhibitors are good examples of down-modulation of function through covalent steric hindrance or modification. Suicide substrates that irreversibly bind to the active site of the enzyme at the catalytically important amino acid of the active site are examples of covalent modifications that sterically block the enzymatic active site. An example of a suicide substrate is TPCK against chymotrypsin (6). This type of modulation, in embodiments of the present invention, covalently binds to a catalytically active site or cleaves a portion from an inactive catalytic site, thereby converting it to a catalytically active one. Can be used for selection of possible compounds.

  There are also examples of non-covalent steric hindrance involving many repressor molecules. Lambda repressors are of interest. This is because it simultaneously down-modulates the expression of other phage genes, such as cro, while up-modulating its own expression. This is because it binds non-covalently to DNA sequences and sterically hinders the interaction of these sequences with RNA polymerase, thereby preventing RNA polymerase from transcribing towards the cro gene. , Achieved by stimulating RNA polymerase to transcribe in the opposite direction. Thus, repressor molecules can cause steric hindrance, and interfere with specific DNA-RNA polymerase interactions to down-modulate the function of DNA sequences.

  Since binding molecules can be made based on their ability to modify the activity of various substrates of interest, the choice of non-covalently binding compounds offers possibilities and advantages.

  The allosteric effect is another way through which modulation is performed in certain biological systems. Aspartate transcarbamoylase is a well-characterized allosteric enzyme. It is the control domain that interacts with the catalytic subunit. Upon binding to CTP or UTP, the regulatory subunit can induce quaternary structural changes in the holoenzyme that down-modulates catalytic activity. In contrast, binding of ATP to its regulatory subunit can up-modulate catalytic activity (7). By using the method of the present invention, it is possible to select molecules that can bind and cause a modulated fourth or third order change.

In addition, various chemical modifications, such as phosphorylation, methylation, acetylation, adenylation and uridenylation can be performed to modulate function. It is known that such modifications play an important role in the control of many important cellular components. The reference ((2), page 73) lists various bacterial enzymes that undergo such modifications. In addition, many proteins associated with human disease are also subject to such chemical modifications. For example, many oncogenes are modified by phosphorylation and have been found to modify other proteins through phosphorylation or dephosphorylation. The ability to select molecules based on their ability to alter the activity of growth factors, for example by phosphorylation, is important.
D. Preferred selection molecules
(1) Fusion or deletion molecules Fusion proteins that introduce growth factors, selection moieties and recognition sequences are preferred selection molecules. A fusion can be between virtually any molecule and can include a fusion between two molecules or multiple molecules. Fusions may include protein-protein fusions and protein-biomolecule fusions. The molecule may be a biological or chemical molecule, or an ion. Sugars, nucleotides, nucleosides, fatty acids, small organic molecules, metal ions such as Mg, and various derivatives and precursors of the foregoing are contemplated. Other fusions may be protein-nucleic acid, protein-ribonucleic acid, protein-lipid, protein-oligosaccharide, nucleic acid-small molecule, small molecule-protein-lipid, nucleic acid-small molecule-lipid, among others.

  If the protease is the molecule to be obtained, a fusion construct of the metabolically important enzyme, i.e., growth factor, with the desired peptide recognition sequence and the bulky protein, i.e., the regulatory moiety, can be made. Steric hindrance and / or conformational changes caused by peptide recognition sequences with bulky proteins inactivate or functionally impair metabolically important enzymes. However, in the presence of the protease to be obtained, the recognition sequence is cleaved and the enzyme function is up-modulated.

  The subunit embodiment and the α-β type complementation embodiment are yet another variation of the fusion construct. The subunit embodiment takes advantage of the complex multi-subunit characteristics of certain molecules. In some cases, the molecule is non-functional as a monomer, but gains function in a multi-subunit complex comprising the same or different groups of molecules. One molecule can be fused with any number of different subunits from another molecule. Due to the subtle and often complex subunit interactions in the active multi-subunit form, the modulation in the functions induced by constructing the fusion tends to be quite powerful.

  The α-β type complementarity aspects are conceptually similar. It is known that certain functional molecules are fragmented and do not function on their own. However, the fragment can recombine and regain function. These fusions are useful in the methods of the invention because these fragments are prevented if these fragments are introduced into the fusion.

  Embodiments of “reverse” subunits can also be used to create fusion constructs. In this case, the multi-subunit complex lacks function and the subunit itself has function. For example, it is possible to create a non-functional multi-subunit string of such proteins using a protein whose function is impaired by the addition of an extra sequence at one of its ends. . Proper design of the linker sequence between the proteins yields a protease with the desired specificity.

  Under strict selection conditions, very few advantages in terms of efficient use of materials can result in differential selection. If one member of the host cell group produced extra protein, it would unnecessarily consume the amino acid pool, penalizing its growth. The use of each subunit portion of the reverse subunit fusion molecule eliminates wasted protein synthesis in host cells that produce novel proteases, so the reverse subunit method is efficient. possible.

  These fusion proteins can take many different configurations, may be active or inactive, and may be up-modulated or down-modulated. These may have protein groups or other groups such as phosphate groups or methyl groups added or deleted internally or at both ends. For example, for a protein growth factor, adding a protein sequence to its interior or one or both ends to regulate its function higher or lower and / or providing a selected molecule with a particular desired recognition sequence it can.

  Multiple different groups can be added. For example, a multiprotein fusion formed by binding a regulatory group causing a great steric hindrance, a recognition sequence, a growth factor, a recognition sequence, and a regulatory group causing a big steric hindrance in this order can be used.

Alternatively, deletions can be used to construct modulated growth factors. For example, a protein growth factor whose presence has a region that is important for the proper function of the growth factor can be used as a growth factor modulated in a truncated or deleted form lacking this region. In another example, a protein that is phosphorylated in its active form can be used as a growth factor modulated in its non-phosphorylated state. In both cases, normal molecules can be selected that introduce moieties into the modulated growth factor.
(2) Allosteric platform The selected molecule used to obtain a new molecule based on its binding ability can be designed in many ways. A selection molecule based on simple binding may have its function modulated based on the binding of a novel molecule to a recognition sequence on the selection molecule. For example, the novel molecule can bind and / or sterically inhibit the active site of an enzyme-based selection molecule.

  A selected molecule that undergoes a conformational change when bound can be referred to as an allosteric platform. Growth factor function can be modulated in an allosteric manner. Therefore, if the recognition sequence becomes covalently or non-covalently bound, the function of the proliferating moiety changes. The binding domain on the recognition sequence can be varied to accommodate the selection of a wide array of different ligands. An allosteric platform can be a single molecule or many molecules and can be used in any of the methods of the invention.

  For example, one part of the allosteric platform can serve as a receptor for binding a novel molecule and the other part can serve to bind the allosteric platform to a growth factor. In binding the new molecule to the first part, allosteric changes in the growth factor will result in up-modulation or down-modulation of function. The change in function can then be used to obtain new molecules by the methods described elsewhere.

The new molecule to be obtained need not actually bind the selected molecule and the function of the new molecule to be selected need not be binding. For example, selected molecules based on binding can be used to select new molecules with virtually any catalytic activity. By using a selected molecule based on a bond that recognizes the product of the reaction catalyzed by the desired new molecule, the new molecule can be selected without itself interacting with the selected molecule. .
II. Selection method
A. General The selection techniques used in the isolation, manufacture and directed evolution methods of the present invention may be any that have been used in the art, as well as new environments, conditions, procedures and selection pressures. May be used. Efforts should be made to limit mutations in genes that express growth factors, recognition sequences, regulatory moieties or selectable molecules so that a gene encoding a new molecule specific for the original desired recognition sequence must be obtained. .

  This is also true in the isolation, production and directed evolution methods of the present invention. In directed evolution, a rationally designed choice that exposes external influences, such as radiation and mutagenic chemicals, or gives a putative new group of evolution directions By providing conditions, it is desirable to mutate genes with evolutionary ability to encode new molecules. Such methods are more fully described elsewhere.

  Negative or positive selection methods can be used. In a positive selection, a host having a desired function or a member of a group of replicators has a selectable growth advantage. The opposite is true for negative selections. A host or member of a group of replicators having a desired function is one that has a selectable growth disadvantage. In negative selection, the gene of interest expresses a molecule that interacts with the selection molecule or recognition sequence to inhibit growth. If a group of members is capable of growth, a compound that can be killed or otherwise weakened, such as some antibiotics, can be administered to the group. Such techniques can be repeated by known cyclic methods to enrich the desired non-proliferative members of the group.

  It is important that the selection conditions are directed to the specific selection characteristics of the object and that the selection procedure is optimized. For example, it may be beneficial to apply the selective pressure in a cyclic manner and circulate between the high and low selective pressures, or use different conditions to select the same characteristic.

  Different selection molecules and selection methods can be used in different environments. For example, in the case of a gene isolation method in which a gene encoding a novel molecule having a desired property is considered to exist in an existing gene pool, one time selection or one time selection is performed. Iterating (batch selection) is appropriate to achieve isolation. Under such circumstances, it is desirable to use a growth factor that is absolutely essential for growth and that substantially or completely loses its function (maximum modulation of function) when introduced into the selection construct. The desired novel molecule or a very closely related novel molecule is considered to be present in the existing heterogeneous group, and only one or very small number of related clones are selected. For this reason, very severe selection pressure is applied to the system.

  Many generation selection methods are used when the desired novel molecule is not considered to be in the group and needs to be evolved. In some cases, growth factors that are absolutely essential for growth, but whose function is only partially modulated, or that are not absolutely essential, give growth benefits and are fully modulated It is desirable to use growth factors that can be made. This provides for growth, during which the genes that express the new molecule evolve, yielding molecules with properties closely related to the properties of the desired new molecule, and gaining the advantage of selective growth.

Differences in the severity of the selection pressure through the selection molecule design that varies for the different starting groups of new molecules can also be caused by changes in environmental conditions. For example, when the same selection molecule is used, different levels of selection pressure are generated simply by changing the medium, temperature, pH, and the like.
In some such examples, the influence of environmental conditions supplements the function of the selected molecule, and the differential selective pressure is set by changing these environmental conditions.

  Another method in which the selection molecule is used as a “riostat” of selection involves changing the expression level of the selection molecule. Simply increasing the expression level of the selection molecule via an inducible promoter can increase the background in some cases and thus reduce the selection pressure.

Two important techniques of the invention that are common to the aspects of isolation, manufacture and directed evolution are the use of selective molecules that overlap with the control of host cell mutations.
(1) Restriction of undesired mutations In order to maintain a constant target for interaction with new molecules, it affects the fidelity of expression of growth factors and / or recognition sequences and / or regulatory moieties and / or select molecules. It is important to control the host cell mutations provided. It is also important to limit background mutations that can complicate selection for the desired novel molecule, for example mutations in DNA throughout the selection system. Unuseful mutations of the gene that expresses the selection molecule or its components include those that confer growth factor function through means other than the desired interaction of the novel molecule, such as proteolytic cleavage by the novel protease. Such mutations include, among other things, mutations in the growth factor portion of the fusion that counteract the effects of bulky groups, such as bulky group mutations that render the bulky group ineffective in modulating growth factor function, i.e., desirable. A transgene or recombination of a gene encoding a sequence for a growth factor that allows for the expression of a growth factor that is not so modulated, mutations that generate a promoter before the growth factor (point mutations and insertions), ie not usable Development of the ability of post-translational or post-transcriptional modification of selected molecules, such as isolating component parts in such a way that liberates growth factors, and mutations that produce alternative pathways or mutant molecules that perform the same function as growth factors including.

  What is important is the specific mutations necessary to produce the desired new molecule and the specifics that lead to specific insertions or deletions that give rise to an unusable species increase compared to the number and rate of overall mutations The number and rate of mutations and the number and rate of overall mutations. In directed evolution, the number of specific mutations required to generate a gene that expresses a useful new molecule is as low as possible, and the mutation rate gives rise to mutants that have no usefulness. And it is important that it is reasonably high for the rate of mutation. If the probability of generating a non-useful gene mutation is 100 times the chance of generating a useful gene mutation, 100 experiments will yield a gene that expresses a useful new molecule. Although possible, this is far from ideal.

For certain genes, the deletion rate may be as high as ^10-4 . This depends inter alia on the specific gene sequence and the surrounding sequence and the resulting secondary structure. The strong potential of deletion mutations requires careful design that prevents the opportunity to give rise to unusable mutants. These opportunities include Rec A ^- or similar mutations that can reduce the frequency of homologous recombination by a factor of 1000 by appropriate selection of gene sequence and secondary structure (both in the construct and locally). Can be minimized.

  In addition, high fidelity anti-mutagenesis gene replication mechanisms can be used to reduce the frequency of mutations. It is known that certain polymerases can be mutated and thereby replicate with higher fidelity than wild-type polymerases. Such polymerases can be used to reduce the error rate during replication. Mutations can be controlled by keeping the number of replicas low or preventing further replication if the cell cycle is suppressed.

In certain embodiments, evolutionary resetting of a selection molecule is simplified by distinguishing genes that encode the selection molecule. For example, if the selection molecule is encoded in the host cell and the new molecule is encoded by the replicator, a selection procedure is possible that evolves the gene encoded in the replicator. This evolved group can then be used in a selection procedure that uses a new group of host cells from the initial starting culture. The components of the selection system encoded by the host cell, for which mutations are undesirable, are evolutionarily reset, while the evolutionary progress of the new molecules can be maintained and continued in a static selection environment.
(2) Use of multiple overlapping selection molecules Another way to enhance the selection of undesired variants of genes expressing new molecules is through the simultaneous use of multiple overlapping selection molecules. . In such a system, a single undesired variant will not cause undesired selection due to the presence of other selection molecules. Appropriate novel molecule expression affects all selected molecules and allows useful selections to occur.

  For example, a series of different protein-protein-protein fusions are generated, each having the same proteolytic cleavage site and different but essential growth factors. Mutations that release one growth factor from the fusion and change its function do not cause unwanted selection and result in marginal selection because other selection molecules are not affected. If a new molecule with the appropriate proteolytic properties is generated, each of the proteolytic cleavage sites is cleaved and the respective growth factor is released from its respective fusion and selection occurs.

  The level of overlap is also used to modulate the selection pressure. Through different levels of overlap, the total number of cuts required for the production of a given species can be varied. For example, conjugating seven different growth factors to seven different overlapping selection molecules will require more cleavage than using three of these seven components.

  More important is the type of ingredient. For example, certain components in the bacteriophage replication process are catalytic and require a relatively low total copy number to accomplish their work. Other components are not catalytic and require more cleavage. Examples of such components are head and tail proteins, which are structural genes related to virion function. If the head and tail proteins are conjugated to a select molecule, an enormous number of cleavages will be required since each protein of the assembled viral coat must be cleaved. This type of selection construct will place a higher burden on the new molecule as it requires higher rotation to produce a significant amount of viral progeny.

  Two important aspects of the present invention, common to manufacturing and particularly directed evolutionary aspects, are the distinction between the gene encoding the selected molecule and the gene encoding the putative novel molecule, the latter controlled mutation And using some evolutionary starting points for genes that express putative novel molecules.

(3) Separation of genes encoding selected molecules and putative novel molecules, and controlled mutation of genes encoding putative novel molecules Genes encoding putative novel molecules and Taking a selection method that separates both genes, for example in time or space, or by different recognition sequences, so that the genes encoding the selection molecules are each replicated by different replication systems (which have different mutation rates) desirable. This allows a potential new molecular gene pool to develop or evolve at an increased rate. While evolving genes that can code for new molecules, the stability of the selected molecule is maintained.

  A gene encoding a putative novel molecule is initially a gene that may be mutated and evolved into a gene that expresses a novel molecule with the desired function, or a gene population Can be mutated and evolved to obtain a gene that expresses a molecule having the desired function. The starting molecular population may or may not be related to the new molecule that is ultimately acquired structurally or otherwise.

  The methods of the invention can be performed by utilizing a low fidelity replication mechanism, by using different types of coding material, and by employing various process conditions that regulate the mutation rate. Also, sequence repair mechanisms that act differentially on genes that express putative novel and selected molecules or their components may be utilized. Also, applying different environmental conditions such as temperature, pressure, pH, ion and substrate concentration effects, etc. can be utilized to achieve these objectives. A regulated mutation rate can also be created by using certain coding substances, such as known “hot spots” in the chromosome, where the mutation rate is higher than elsewhere. it can.

  Physically distinct coding sequences with different mutation rates and / or different replication mechanisms are used. These include in particular host chromosomal DNA, plasmid DNA (circular or self-replicating short sequences), viruses (both DNA and RNA), short self-replicating RNA sequences or plasmids, and mitochondrial DNA. These and other coding sequences can be used in any combination to encode selected molecules and putative novel molecules. These different code carriers can replicate at different mutation rates by using different replication mechanisms with different specific origins of replication.

  Another way in which differential mutation rates can occur is by replication timing and turnover (turnover). For example, if replication occurs at a faster rate in a sequence encoding a new molecule than in a sequence encoding a selected molecule (both using the same fidelity replication mechanism), the population of new molecules will have a large number of mutations. Will develop. In addition, the timing of synthesis of different components into different replication mechanisms can be used to produce different mutation rates. For example, the E. coli chromosome is replicated in a controlled manner, and certain proteins, such as DnaA, are known to be required for replication initiation. It is therefore possible to have a system in which two DNA polymerases with different mutation rates are controlled by an inducible promoter or a cyclically activated promoter. When synthesizing polymerase and DnaA type proteins, high fidelity polymerases, for example, regulate the timing so that the selected molecule replicates on the host chromosome and the low fidelity polymerase replicates the new molecule on the viral chromosome it can. In some embodiments, such systems can have the same origin of replication.

(4) Number of evolutionary start points and mutations in gene populations Several different methods can be used to create populations of genes that express novel molecules. Zero evolution start points, one evolution start point or multiple evolution start points may be used.

  A selection system designed to have no evolutionary starting point can be used to select a gene of interest based solely on a selection molecule introduced into the host. Such methods select for naturally occurring genes within the components of the selection system, for example genes from the host cell or replicator. In this way, rather than introducing one or more specific molecules that are used as starting points for evolution, a unique evolutionary potential is derived within the host or replicator. An alternative method using a zero evolutionary starting point involves introducing a population of molecules that are all or partly probabilistically generated and thus unknown to the prior art. In this case, the evolutionary potential of these stochastically generated molecules is used to produce genes that express new molecules.

  Two examples of how to use a single starting point of evolution are when a foreign gene is selected and introduced into a host or replicator based on the evolution potential of that gene, and the gene is a member of a subspecies of that gene, For example, when selecting together with its closely related variants.

  Finally, multiple evolution starting points can be used. In this case, a large number of genes are selected based on their evolution potential. These genes can be mutagenized to diversify their subspecies. This enables the creation of new gene populations that are heterogeneous but at the same time rationally designed populations that are highly concentrated based on the desired evolutionary potential.

  As an example, in the development of a catalyst with a particular reaction type and specificity, an evolution starting point is selected that appears to have the highest evolution potential to yield the desired catalyst. A gene is selected that expresses a molecule that already has characteristics similar to those of the desired catalyst, eg, one or more molecules that have specificity for a recognition sequence similar to the desired catalyst. Also, one or more molecules of the same or similar reaction type (eg, proteolysis, hydroxylation, etc.) as the desired catalyst reaction type are selected. By making a reasonable choice of the type of evolution starting point, a large evolutionary potential for the gene population is obtained, and thus a short evolutionary distance to reach the desired gene, i.e., necessary to reach the desired gene. Less mutations are made. Suitable starting points for evolution are enzymes, antibodies, catalytic antibodies, T cell receptors and MHC molecules. Different methods of mutation exist and can be used alone or in combination with each other. One method is the use of conditions such as mutagenic compounds or chemical mutagens or UV irradiation. Site-directed mutagenesis techniques can also be used. A method for site-directed mutagenesis is described in (1). Other methods of increasing the number of mutations in the population include the use of low fidelity replication mechanisms and high replication rates.

  In other techniques, a short stochastic sequence of DNA can be introduced near the coding region of the active site of the enzyme and the population is exposed to drugs and / or UV radiation. This population can then be replicated frequently with a low fidelity replication mechanism to quickly obtain a highly heterogeneous population.

  In other examples, the novel molecule is encoded on the viral genome, and a large population of such viruses has been subjected to one or more mutagens or mutagenesis conditions, such as in vitro chemical Mutagenesis, site-directed mutagenesis, recombination, translocation, UV or light-induced mutation, PCR-mediated mutation, stochastically generated mutation (described in British patent specification 2136661A) Mutation), low fidelity replication, and high replication frequency, but are not limited to these. As a result, the viral population encodes a number of different novel molecules.

  In other examples, non-lytic phages, such as M13 encoding a novel molecule, are replicated with a low fidelity polymerase while DnaA is not produced. The expression of the low fidelity polymerase is stopped and the high fidelity polymerase and DnaA are activated and the E. coli chromosome encoding the fusion is replicated as well. This process is repeated cyclically to continuously promote high mutation rates in the new molecular population.

(5) Other methods of regulating selection pressure to promote directed evolution.Also, the genes encoding a population of new molecules can be selected between high and low selection conditions, or even selection conditions and gene evolution. It is beneficial to cycle between conditions that allow to promote. For example, modified viruses are cycled and incubated between two strains of E. coli, a high selective pressure strain and a low selective pressure strain, resulting in low specificity or low turnover novel molecules with marginal growth Is produced in a high selective pressure strain, the population is increased in the low selective pressure strain, mutagenized, and then again subjected to a high selective pressure. This cycle is repeated to facilitate the evolution of the new molecular population for the desired specificity.

  Depending on the stability of the high selective pressure strain and the mutation rate of the selected molecule, a continuous or semi-continuous system will occur. In a continuous system, the modified virus is continuously produced in a low selective pressure strain. Partial reintroduction into the low selective pressure incubation chamber so that free viral particles are recovered, mutagenized, and increase the heterogeneity of the population of new molecules beyond the level that can only occur with low fidelity replication Is done. The remainder is incubated with a high selective pressure strain. Low selective pressure strain incubation chambers are inoculated periodically with new bacteria, or the conditions are adjusted so that the replication rate of the strain relative to the replication rate of the virus is sufficient to maintain the bacterial population.

  In certain embodiments where growth factors essential for viral replication are down-modulated, high selective pressure bacteria in a continuous system need to be replenished until a new molecule with some desired specificity is produced. There will be no. This is because infection with a modified virus having a gene encoding a novel molecule that is not capable of cleaving the selection molecule is unsuccessful. It is only when the selection construct is cleaved and the function that the infection proceeds through the lysis cycle is regained in the viral replication machinery. Once a new molecule with some ability of the desired molecule is produced, viral growth is promoted. Selection is continued for the desired specificity, reaction type and turnover rate by continuously adding new high selective pressure bacteria to the incubation chamber. The added high selective pressure bacteria are grown directly from the aliquot of the first high selective pressure bacteria, resulting in an evolutionary reset of the bacterial population. The selection method can also use undissolved phage.

  With respect to directed evolution, a population of novel molecules is based on the sequence of molecules, such as proteases, that have properties most similar to the properties of the desired novel molecule, such as activity, specificity, turnover, etc. In certain embodiments, mutagenesis and low fidelity replication are important in generating a stable heterogeneous population, since the gene population encoding the new molecule can be derived from a single source. In addition, the selection pressure can be tuned and / or cyclic to aid in the evolution of the gene encoding the desired novel molecule. Other techniques important to promote directed evolution include increasing the total number of copies of the gene that encodes the population and the heterogeneity of the population of new molecules by using mutator low fidelity polymerases Is to increase.

  For example, the starting point of evolution of a new molecule may be a special protease that recognizes a specific sequence that is not harmful to the system. However, in certain embodiments, it is advantageous to have a plurality of new molecule starting points. Thus, several different proteases of the same family or different families may first be encoded in the modified virus. Where multiple novel molecule starting points are used, starting points that approximate the desired catalytic activity or specificity, or both, may be incorporated.

B. Host selection method
(1) Positive selection Positive cell selection can occur in various ways. In one example, a cell growth factor that provides a cell with a growth benefit is functionally down-modulated. The down-modulated cell growth factor is complexed in the selected molecule, so that the growth factor is functionally up-modulated after production of the desired new molecule. Thus, selection of the desired new molecule occurs by positive cell selection.

  A toxin is also incorporated into the selected molecule, so that its function is maintained or up-modulated. Toxins that are functionally maintained or up-modulated are complexed in the selected molecule, so that, after production of the desired new molecule, the growth factor is functionally down-modulated. The selection of the desired new molecule occurs again by positive cell selection.

  Such positive cell selection techniques can be performed in a variety of different devices, including chemostats and turbidostats, under appropriate conditions, etc. in a simple incubation chamber with the appropriate medium added.

(2) Negative selection Negative cell selection can occur in various ways. A cell growth factor that confers a growth benefit on a cell has its function maintained or up-modulated when complexed in a selected molecule. After production of the desired new molecule and its interaction with the selected molecule, the growth factor is functionally down-modulated. Thus, selection of the desired new molecule occurs by negative cell selection.

  In other examples, the toxin is incorporated into the selection molecule so that its function is down-modulated. However, the toxin is functionally up-modulated after production of the desired new molecule. Again, selection of the desired new molecule occurs by negative cell selection.

  Negative cell selection is performed using methods such as growth in the presence of molecules such as antibiotics that preferentially kill the proliferating cells. Cells capable of growth can be selected by cycling the cells from a medium containing antibiotics through a growth medium that does not contain antibiotics.

C. Replicator selection method
(1) Positive replicator selection In an embodiment of the invention that utilizes positive selection of a replicator, e.g., a virus, the gene to be isolated, generated or evolved and its gene pool or its mutant Coded within the population. The viral population is made to infect host cells. Viral particles with genes that express novel molecules are given preferential growth advantages (lysis pathway) or integration advantages (lysogen pathway). Where viral replication is a selectable property, selection molecules may be made using essential proteins involved in viral replication that are functionally down-modulated in the selection molecule. A gene encoding the novel molecule is then obtained based on its interaction with the recognition sequence and up-modulation of growth factors and the resulting viral replication.

  Positive virus selection provides many inherent advantages. Viruses are highly efficient carriers of myriad coding sequences and provide a convenient way to physically separate the coding material. In addition, some viruses encode their own replication machinery and origin of replication, allowing high fidelity and low fidelity replication systems to work simultaneously (one for the virus and one for the host). Work against). Viruses also replicate at a faster rate than bacteria. This allows a large difference in the number of replicas per unit time between the host and the virus, which can result in a difference in the total number of mutations. Therefore, it promotes positive selection and variability, i.e. high mutation rate in the population encoding the novel molecule, and homogeneity, i.e. low mutation rate in the population encoding the selected molecule, thereby non-beneficial variation It is possible to minimize body selection.

  The method of the present invention utilizing positive selection of the virus and negative selection of the host cell can be performed by generating a modified bacteriophage encoding a novel molecule and a first modified strain of E. coli encoding a selection molecule. . A modified phage does not encode all of the components necessary for its replication. Those replication components that are not encoded in the modified phage are encoded in the first modified E. coli strain in a selection molecule that modulates their function.

  Modified phage encoding a putative novel molecule are produced in a second modified strain of E. coli prior to selection by the first strain. A modified bacteriophage that lacks one or more components important for replication will not replicate in a typical host cell, thus encoding and producing one or more components that are missing in the modified phage. Two strains are formed to produce a large population of modified phage for selection by the first strain.

  The selective pressure may be applied against the virus in a continuous or semi-continuous flow cell culture, and a normal virus assay may be performed. If conditions and / or dilutions are appropriate, negative selection of cells can also be used. In this case, a virus having a gene that expresses a molecule to be isolated, created or evolved has a selective advantage in the host cell that can give the host cell a selective disadvantage. The method described for negative selection can then be used to select infected cells.

(2) Selection of a negative replicator by positive selection of the host The selection of the gene of interest can be performed by negative selection of replicator growth and / or replication and / or positive selection of host cell growth. Lysed viruses give the virus a growth benefit when complexed in the selected molecule, but do not give the virus when the desired new molecule is expressed and interacts with the selected molecule to release growth factors. It can be used with host cells that encode viral growth factors that confer a selective disadvantage. Such a selection molecule may be an enzyme essential for viral replication having a recognition sequence inserted into the enzyme that does not downmodulate the activity of the enzyme. After interaction with the desired new molecule and cleavage of the recognition sequence, the viral replicase becomes functionally down-modulated. The virus is negatively selected and the host cell receives a positive selection.

D. Multiple Replicator Methods Multiple replicators may also be used in the same host, ie different components of the selection method may be encoded by different populations of replicators. For example, different replicators may encode one or more of a putative new population of molecules, recognition sequences, modulating moieties, selected molecules, and the like. Different viruses and plasmids may be used in combination with each other, resulting in a heterogeneous population of genes encoding the novel molecule.

Such a method for the isolation, creation or directed evolution of a gene encoding a novel molecule capable of the desired interaction with the substrate of interest includes:
In one or more populations of replicators in a host cell population, multiple copies of one putative novel molecule or multiple putative novel molecules, for one or more replicator populations that express the novel molecule Expressing a growth factor and a substrate of interest or analog thereof functionally related to said growth factor, and optionally a modulating portion of said replicator growth factor and imposing selection conditions, for example, host cells And incubating the population of cells under selective conditions to select a replicator that expresses a novel molecule that interacts with the analog to alter the activity of the growth factor.

  The order of expression of some components is a matter of choice, such as relative timing of expression and imposition of selection conditions. It is also possible to exogenously add one or more of the growth factors, recognition sequences or modulating moieties, as in another embodiment of the invention.

E. Selective selection methods based on diseased cells can also be performed directly in diseased cells. For example, cancer cells can be used as a host that encodes a selection molecule, which allows selection of new molecules (which slow the growth of cancer cells). As an example, cancer cells that unduly phosphorylate cellular enzymes important in cell cycle regulation, thus resulting in a cancer phenotype, can be used as hosts. Selection is done for new molecules that can reverse the cancer phenotype. The new molecules thus selected are then analyzed in normal cells of the same type as cancer cells for toxic or harmful side effects. The methods of the invention can be used in cells of a disease state to select molecules that reverse the disease state to a normal phenotype.

F. Selective antibodies (or fragments thereof) based on the recognition of products produced by the interaction of new molecules have a high degree of specificity and can elucidate very slight differences in molecular structure, In fact, it is possible to specifically recognize molecules. Other binding molecules include T cell receptors, MHC molecules and lectins.

  The ability of antibodies to bind antigens in a highly specific manner is well documented, for example, the isolation, creation or orientation of site-specific hydroxylases for the conversion of the drug RH to ROH (8) It can be advantageously used to carry out the method of the invention in terms of evolution. An advantageous method is described in FIG. 3 and detailed below.

  First, in this example, a small amount of desired product or by synthesis / isolation or chemical synthesis, such that a monoclonal antibody specific for the hydroxylated drug ROH can be produced by a protocol such as the protocol of (9). It is necessary to obtain analogs of this product. The antibody is then expressed in its functional conformation in E. coli by extending the techniques of (10), (11), and (12). This recombinant antibody is further developed as described below to link its binding of ROH to cell proliferation.

  An important property of several classes of antibodies is that the constant region of the antibody undergoes a conformational change after binding. This conformational change is evident, for example, with IgM class antibodies. Frank et al., In their chapter in the appendix to (13), state that “[antigen] binding promotes conformational changes in antibodies”. Such conformational changes are further described in (14), (15), and semi-cited in (15) and described in (16) and (17). In addition, it is known that conformational changes can contribute to antibody binding (18). These conformational changes can be used to link product formation and subsequent antibody binding to growth modulation. This tie in to growth rate modulation can be made in a variety of different ways. Proteins in which the function of the protein is highly modulated based on the conformation of the bound constant region (ie, bound or unbound) can be used. Alternatively, in a preferred example, tying to cell proliferation is by binding proteins that are modulated based on whether function is covalently bound to a constant region. Such proteins are proteins that are only active in multi-subunit holoenzymes, or peptide sequences that are only active when they are not sterically hindered, or sequences that are active in areas that are inaccessible to large antibodies. There may be. Examples are multi-subunit aspartate transcarbamoylase, a gene essential for pyrimidine nucleotide synthesis. This enzyme contains 12 polypeptide chains and its catalytic activity is modulated by conformational changes (19). Enzymes such as aspartate transcarbamoylase, which are highly complex and whose catalytic activity is highly sensitive to conformation, are good candidates. This is because their function can be modulated when their subunits are conjugated to large antibody molecules. In addition, in aspartate transcarbamoylase, the active site is formed at the interface of two subunits. A method for linking the aspartate transcarbamoylase subunit gene to the constant region of the recombinant antibody is described in Example I.

  In order to obtain new molecules capable of producing the desired specifically hydroxylated drug ROH, cell selection under pyrimidine nucleotide-restricted conditions can be performed using host strains (which can function aspartate transcarbamoylase for effective growth. Required and lacking non-specific proteases). Cleavage of aspartate transcarbamoylase from the constant region of the antibody occurs when the antibody binds to ROH. This is accomplished by the use of a protease that is specific for the constant region conformation of the antibody when the antibody is bound to its antigen. Specific proteases are developed using the techniques of the present invention. Aspartate transcarbamoylase is cleaved from the selected molecule and forms its active multi-subunit form when the antibody is bound to its antigen catalyzed by the desired hydroxylase, ie, the drug, ROH.

  Conformational changes induced by binding can be approached in different ways. Dense packing of antibodies after cross-linking or ligation by antigen grouping can also cause conformational changes. The closely packed quaternary or tertiary structure of the antibody can be targeted. In related methods where dense packing of antibodies usually does not occur, a dummy antibody that binds to an epitope other than the desired epitope but is spatially close to the desired epitope can be used. Thus, a tightly packed conformational change of the antibody will occur after binding of the antibody to the desired epitope.

  Embodiments of the invention based on the recognition of products produced by the interaction of novel molecules include, among other things, the selection of novel kinases, phosphatases and methylases or molecules that can be uridinylated, adenylated, hydroxylated or glycosylated.

G. Modulating the level of activity of a novel molecule by modulating the expression level of the putative novel molecule The selection pressure of a novel molecule can be adjusted by setting the level of expression of the putative novel molecule. When trying to develop an enzyme with a desired function, the putative level of expression of the new molecule can be used to direct the desired turnover rate of the new molecule. If a high turnover rate is selected, the expression of the putative new molecule is regulated at a low level. Also, if a low turnover rate is desired, the expression of a putative new molecule is regulated at a high level. More particularly, if a high turnover rate is desired, a large number of new molecules present in the system at any one point in time (and considering their synthesis rate and degradation rate) Selective molecules can be used that ensure that new molecular reaction events are required per unit time to provide selectivity. Thus, the selection pressure is
It can be added by appropriately setting the number of putative new molecules present at any given time.

H. Selection of certain proteases The host cell or replicator selection method of the present invention can be used to isolate, create or direct the evolution of novel proteases that can cleave certain substrates of therapeutic interest. Can be done.

An example of such a recognition sequence is an influenza hemagglutinin epitope. In particular, the following sequences can be incorporated into the selected molecule:
Site A amino acids 140-146,
Lys-Arg-Gly-Pro-Gly-Ser-Gly or
Lys-Arg-Gly-Pro-Asp-Ser-Gly or
Lys-Arg-Gly-Pro-Asp-Asn-Gly, or site B amino acids 187-196
Thr-Asp-Gln-Glu-Gln-Thr-Ser-Leu-Tyr-Val or
Thr-Asn-Gln-Glu-Gln-Thr-Ser-Leu-Tyr-Val or
Thr-Asn-Lys-Glu-Gln-Thr-Asn-Leu-Tyr-Val, or site C amino acids 273-279
Pro-Ile-Asp-Thr-Cys-Ile-Ser or
Pro-Ile-Gly-Thr-Cys-Ile-Ser or
Pro-Ile-Asp-Thr-Cys-Ser-Ser or amino acids 52-54
Cys-Asn-Asn or
Cys-Asp-Asn or
Cys-Asn-Lys

Other examples of such recognition sites are the following sites of HIV gp120:
(a) Variable region 3, amino acids 271-295
NNTRKSIRIQRGPGRAFVTIG-KIGN
(b) Conserved domain 4, amino acids 392-402
QFINMWQEVGK
(c) Conserved domain 5, amino acids 452-474
ELYKYKVVKIEPLGVAPTKAK-RR

  Proteases can be produced for such sequence variants. In addition, overlapping selection molecules having the same basic conformation of such sequences can generally be used to select proteases that are specific for their entire conformation.

I. Use of a combination of a recognition sequence and a gene that expresses a putative novel molecule A combination of a recognition sequence and a gene that expresses a putative novel molecule may be used. Two different recognition sequences can be used in each of the two overlapping sets of selected molecules along with genes that express two putative novel molecular populations that are distinguished by their evolutionary origin. A new molecule is obtained from a first population that reacts with one of the recognition sequences, and a new molecule is obtained from another population that reacts with other recognition sequences. In other examples, a selection molecule is used that incorporates two closely related recognition sequences, A and A ′. Interaction with both recognition sequences by the desired novel molecule is required to give some degree of selectivity. Selection can be made for reactivity to both recognition sequences simultaneously. This can occur by selection of one molecule with broad specificity for A and A ′, or selection of two molecules, one with specificity for A and the other with specificity for A ′. . In each case, reactivity to both A and A ′ is selected simultaneously. In a similar manner, multiple selection molecules can be used in which one set of selection molecules contains A and the other set contains A ′ in the overlapping selection molecule.

J. Cell-Free Method The present invention can also be performed in a cell-free method. A preferred embodiment of the cell-free method is disclosed in FIGS. 4A and 4B.

  Referring to FIGS. 4A and 4B, reference numeral 200 represents multiple copies of a putative novel molecule or DNA encoding a number of different putative novel molecules. DNA 200 is mutagenized, thereby forming a heterogeneous population 202 of DNA that encodes a number of different putative novel molecules. Population 202 is transcribed and translated to express a number of different putative novel molecules 204. Reference numeral 206 represents the novel molecule of interest within the population.

  Reference numeral 208 represents actin as a growth-modulating moiety and DNA encoding a select molecule comprising a recognition sequence that is or represents a substrate of interest. DNA population 208 is transcribed and translated to express a population of selection molecules 210.

  The putative new molecule population 204 is then incubated with the population of selected molecules 210 in the incubator 212. Incubation of the putative new molecule and the selected molecule results in enzymatic cleavage of the recognition site within the selected molecule by the desired new molecule 206, thereby releasing the actin monomer 214 from the selected molecule. After the reaction is complete, DNase 216 is added to the reaction mixture, and the mixture is incubated in incubator 218 for a time sufficient to allow free actin, if present, to inhibit DNase. . Excess incubated mixture from incubator 212 is used for DNase 216 so that all of the DNase is inhibited. The actin monomer inhibits DNase as indicated by reference numeral 220. Next, after actin is inhibited, a heterogeneous population of mutated DNA encoding a population of different putative novel molecules 202 is transformed into a number of different putative novel molecules, actin-based selection molecules and DNase incubated mixtures. And the mixture is further incubated in incubator 218. The presence of the desired new molecule is analyzed by the presence of non-degraded DNA.

  DNA is isolated / purified, distributed, expressed, and reanalyzed for the desired function of the new molecule. In a preferred method, isolated / purified DNA is amplified, for example, by polymerase chain reaction (PCR), and the amplified DNA is subjected to one or more iterations of the method to select the desired DNA.

  The invention is further illustrated by the following examples.

Example I
Generation of novel one or more proteases of decapeptide sequence from HIV gp 120 by using artificial zymogen and positive selection of virus Example I shows the use of one or more endopeptidases specific for the decapeptide recognition sequence from gp120. Describe how to make. The method uses gene fusions that encode protein-based selection molecules. A novel protease encoded in the viral population cleaves the decapeptide recognition sequence, thereby releasing the proteins necessary for viral replication. This example is representative of positive selection of viruses. Simplified schematics are shown in FIGS. 2A, 2B and 2C.

  E. coli B, the host strain of bacteriophage T7, is transformed by introduction of a plasmid to complement the T7 deletion mutant. Two different deletion mutant T7 strains are generated and thus two corresponding E. coli complementary transformants are generated as listed below.

  1) E. coli transformant 1 (ET1), T7 gene gp1 (RNA polymerase), gp4 (primase / helicase), and mutants with low fidelity and reduced 3'-5 'exonuclease activity It is transformed with the E. coli plasmid pKK177-3 having the sequence for inducible expression of gp5 (DNA polymerase). This transformed host allows for the growth of T7 deletion mutants (T7A) of genes gp 1, 4, and 5 by inducible expression of these genes.

  2) E. coli transformant 2 (ET2) was transformed into T7 gene gp1, gp4, mutant gp5 with low fidelity and reduced 3'-5 'exonuclease activity, and in addition gene 10A (major head protein ), 13 (internal virion protein), and 18 (DNA mature protein) with E. coli plasmid pKK177-3 having sequences for inducible expression. This transformed host allows for the growth of T7 deletion mutants (T7B) of genes gp 1, 4, 5, 10A, 13, and 18 by inducible expression of these genes.

  In addition, two E. coli selection transformants (EST1 and EST2) are prepared. These transformed cell lines differ from ET1 and ET2 in that the T7 gene encoded in these cells is complexed as a gene fusion. In addition, these strains are selected for protease deletions (eg, strains such as lon, hfl, or htpR) (1) and (20). These two transformants are described below.

  1) Select E. coli selective transformant 1 (EST1) for the expression of T7 genes gp1, gp4, and mutant gp5 with low fidelity and reduced 3'-5 'exonuclease activity (these All of the sequences have modulation moieties (virus particle structure coat protein from T4) fused to both their amino and carboxy termini as outlined in the table below linked by a gp120 protease recognition sequence) E. coli plasmid pKK177-3 having

2) E. coli selective transformant 2 (EST2) was transformed into T7 gene gp1, gp4, mutant gp5 with low fidelity and reduced 3'-5 'exonuclease activity, and in addition gene 10A (major head Protein), 13 (internal virion protein), and 18 (DNA mature protein) sequences for inducible expression (all these sequences are those as outlined in the table below linked by the gp120 protease recognition sequence) Is transformed with the E. coli plasmid pKK177-3 having a modulating moiety (with a viral particle structure coat protein from T4) fused to both the amino terminus and the carboxy terminus.
T7 gene amino terminal T4 gene carboxy terminal T4 gene
1 7 20
4 27 23
5 14 15
10A 18 12
13 17 24
18 3 hoc

The complete sequence of T7 is described in (21). Using information from the T7 sequence, primers are chemically synthesized for PCR-mediated amplification of the gene to be inserted into the plasmid. Examples of PCR primers for each gene are listed below, and the numbers correspond to the T7 DNA sequence shown in (21).
Gene primer A primer B
1 3171-3185 5806-5820
4 11565-11579 13249-13263
5 14353-14367 16451-16465
10A 22966-22980 23987-24001
13 27306-27320 27706-27720
18 36552-36566 36805-36819

  PCR amplification of each of these genes is performed by conventional techniques such as the technique described in (1). T7 wild type phages and their DNA used as templates are isolated / purified by the following method as described by (22) and (23).

  Once amplified, the T7 gene is ligated to a plasmid vector having an inducible promoter such as p-KK177-3 (the vector uses the tac promoter). The tac promoter is turned off in an E. coli strain that expresses high levels of lac repressor. The promoter is induced by the addition of isopropylthio-β-D-galactoside (IPTG) to a final concentration of 1 mM. The p-KK177-3 and tac promoters are described in (1). Ligation of the T7 gene amplified by PCR to a plasmid containing an inducible promoter such as p-KK177-3 can be performed by the usual ligation technique described in (1). The recombinant plasmid is then used to transform E. coli B strain (which expresses high levels of lac repressor) by techniques such as electrotransformation or by using calcium chloride. Protocols for these technologies are described in (1).

  Gene fusion is commonly used in molecular biology and a variety of different methods for the construction of β-galactosidase fusion proteins are described in (24). Using these methods or the method described in (1) referred to above, form an appropriately linked selection molecule containing the T7 gene with the T4 structure coat protein gene linked to both the amino terminus and the carboxy terminus. As a result, an open reading frame is maintained in the construct. Thus, each of the T7 genes in EST1 and EST2 encode without a stop codon that terminates the fusion protein early or an inserted or deleted base that shifts the reading frame. The T4 DNA sequence may be placed and isolated / purified using the T4 genomic map reproduced in (25). The required gene is then PCR amplified for use in ligation procedures as previously described for the T7 gene. The DNA molecule encoding the fusion can then be ligated in turn to pKK177-3 plasmid DNA under the control of the tac promoter.

  The protease recognition sequence is a decapeptide near Trp397 in the fourth contact region of HIV virus gp120. This amino acid is very important for the binding of gp120 to CD4. This is because the gp120 mutant at this position terminates CD4 binding. From the sequence of gp120 from (26), the desired proteolytic recognition sequence, for example FINMWQEVGK (phenylalanine-isoleucine-asparagine-methionine-tryptophan-glutamine-glutamic acid-valine-glycine-lysine) can be determined. The sequence encoding the nucleotide is obtained from (27). It describes the nucleotide sequence from five different HIV clones, each of which has Trp397.

  There are at least four different methods that can be used to generate T7 deletion mutants. These methods include the use of specific restriction enzymes where appropriate, creating unique restriction sites by oligonucleotide-mediated mutagenesis, deletion by oligonucleotide-mediated “loop-out” mutagenesis, and strains. Including the occurrence of genetic deletions. These techniques are outlined in (1). The T7 viral genome is obtained as previously described in (22) and (23). Next, the T7 deletion mutant genome is appropriately packed into virus particles in vitro according to the protocol of (28).

  The initial population of new molecules is based on several known proteases. This allows several different starting points of evolution from which the desired new molecule can arise. Proteases are HIV protease, polio 3C protease, and subtilisin BPN '. The sequences of these proteases are introduced into E. coli and expressed as functional enzymes or enzymes that can have zymogen status (29) and (30) functionality.

  Each protease gene is subjected to site-directed mutagenesis as well as in vitro chemical mutagenesis to generate large and heterogeneous putative novel molecular populations. The method of site-directed mutagenesis is shown in (1). The method of in vitro chemical mutagenesis is shown in (1).

  The new mutagenized molecule is then converted to a promoter, eg, bacteriophage lambda PR promoter or PL promoter (for use in the host, these lambda repressors that down regulate the production of the new molecule by the methods described above. Not expressed) or connected to T7 promoter. The promoter / novel molecule population is then linked to the T7A deletion mutant population grown in ET1 host cells according to the procedure described above. Once this operation is completed, a heterogeneous T7A deletion mutant population is produced, which is highly heterogeneous due to the fact that it encodes a heterogeneous mutagenized pool of novel molecules. This population is hereinafter referred to as T7A / novel molecule.

  The T7A / new molecular population is then propagated on ET1 host cells and transferred to EST1 host cells. Keep the EST1 host cells of the T7A / new molecular population in quiescent non-replicating growth phase Only T7A / new molecular virus particles that can produce new molecules that restore gp1, gp4, and gp5 functions by cleaving the gp120 decapeptide protease recognition sequence, then release the T4 virus particle structure coat proteins from the selected molecule Can thus be propagated in these hosts. T7A / novel molecules are added in a continuous manner using a cellstat as described in (31), or repeated aliquots of T7A / novel molecules are added to the EST1 host in a semi-continuous batch process. Once viral replication occurs, the resulting viral particles are recovered and their DNA is isolated / purified as described in (22) and (23). Isolation / purification of T7 / fragment of novel molecular gene that can hybridize with DNA encoding wild-type novel molecule by PCR amplification when primer recognition sequence is not mutated in a suppressive manner Or by isolation / purification of a fragment capable of expressing a molecule having the desired function.

  Next, sequences encoding new molecules from T7 / new molecule virus particles that can be propagated in EST1 are expressed in any of several expression systems (1), and then proteases are more accurately functionally characterized. Can be determined. If further selection is needed, the sequence of the new molecule from the T7A / novel molecule virus particle that can be propagated in EST1 is linked to T7B. Thus, these operations are performed as described above except that T7B is used instead of T7A, ET2 is used instead of ET1, and EST2 is used instead of EST1.

  Thus, the procedure of Example I allows the development of new proteases specific for the decapeptide sequence from gp120.

Example II
Creation of one or more novel proteases for β-galactosidase by use of artificial zymogens Example II is a method for creating one or more endopeptidases with a range of specificities for the recognition sequence near the carboxy terminus of β-galactosidase. Is described. The method uses a gene fusion with β-galactosidase and T7 gene. The selection operation is based on a novel molecule (protease) that cleaves β-galactosidase that upmodulates the T7 product function required for viral replication. Therefore, this example shows a positive selection operation for viruses. The operation is generally shown in FIGS. 2A, 2B and 2C.

  Specifically, β-galactosidase in the proper reading frame is linked to the respective amino terminus of the T7 gene encoded in EST1 and EST2, which does not contain a stop codon that prematurely terminates the fusion protein. The DNA encoding the fusion is then inserted into the pKK 177-3 plasmid under the control of the tac promoter.

  E. coli B, the host strain of bacteriophage T7, is transformed by introducing a plasmid encoding the T7 gene so as to complement the T7 deletion mutant. This allows the growth and amplification of these T7 deletion mutants. Two different deletion mutant T7 strains are generated and thus two corresponding E. coli complementary transformants are produced as described in Example I. In addition, corresponding selection strains that are EST1 and EST2 containing β-galactosidase fused to the amino terminus of the T7 gene used are then used for the selection procedure.

  There is no restriction on the protease other than cleaving enough β-galactosidase to exert the function of the T7 gene in the construct. In this regard, the desired new molecule is “semi-specific”. However, the protease itself may be specific for a particular recognition sequence. One skilled in the art will recognize that this technique can be used to develop proteases with specificity for the recognition sequence using proteins other than β-galactosidase.

  1) E. coli transformant 1 (ET1), T7 gene gp1 (RNA polymerase), gp4 (primase / helicase), and mutant with low fidelity and reduced 3'-5 'exonuclease activity It is transformed with the E. coli plasmid pKK177-3 having the sequence for inducible expression of gp5 (DNA polymerase). This transformed host allows for the growth of T7 deletion mutants (T7A) of genes gp 1, 4, and 5 by inducible expression of these genes.

  2) E. coli transformant 2 (ET2) was transformed into T7 gene gp1, gp4, mutant gp5 with low fidelity and reduced 3'-5 'exonuclease activity, and in addition gene 10A (major head protein ), 13 (internal virion protein), and 18 (DNA mature protein) with E. coli plasmid pKK177-3 having sequences for inducible expression. This transformed host allows for the growth of T7 deletion mutants (T7B) of genes gp 1, 4, 5, 10A, 13, and 18 by inducible expression of these genes.

  In addition, two E. coli selection transformants (EST1 and EST2) are prepared. These transformed cell lines differ from ET1 and ET2 in that the T7 gene encoded in these cells is complexed as a gene fusion. In addition, these strains are selected for protease deletions (eg, strains such as lon, hfl, or htpR) (1) and (20). These two transformants are described below.

  1) The E. coli selection transformant 1 (EST1) was transformed into the T7 gene gp1, gp4 and the sequence of mutant gp5 with low fidelity and reduced 3′-5 ′ exonuclease activity, each of which is β -Ligated to galactosidase, so that expression is transformed with the E. coli plasmid pKK177-3 with the modulating moiety β-galactosidase fused to their amino terminus).

  2) E. coli selective transformant 2 (EST2) was transformed into T7 gene gp1, gp4, mutant gp5 with low fidelity and reduced 3'-5 'exonuclease activity, and in addition gene 10A (major head Protein), 13 (internal virion protein), and 18 (DNA mature protein) sequences, each of which is linked to β-galactosidase, so that expression results in the modulation moiety β-galactosidase fused to their amino terminus. E. coli plasmid pKK177-3 having

  The complete sequence of T7 is described in (21). Information from the T7 sequence can be used to chemically synthesize primers for PCR-mediated amplification of the gene to be inserted into the plasmid. Examples of PCR primers for each gene are listed below, and the numbers correspond to the T7 DNA sequence shown in (21).

Gene primer A primer B
1 3171-3185 5806-5820
4 11565-11579 13249-13263
5 14353-14367 16451-16465
10A 22966-22980 23987-24001
13 27306-27320 27706-27720
18 36552-36566 36805-36819

  PCR amplification of each of these genes is performed by conventional techniques such as the technique described in (1). T7 wild type phage and their DNA used as templates may be isolated / purified by the following method as described by (22) and (23).

  Once amplified, the T7 gene can be ligated to a plasmid vector (the vector uses the tac promoter) with an inducible promoter such as p-KK177-3. The tac promoter is turned off in an E. coli strain that expresses high levels of lac repressor. The promoter is induced by the addition of isopropylthio-β-D-galactoside (IPTG) to a final concentration of 1 mM. The p-KK177-3 and tac promoters are described in (1). Ligation of the T7 gene amplified by PCR to a plasmid containing an inducible promoter such as p-KK177-3 can be performed by the usual ligation technique described in (1). The recombinant plasmid is then used to transform E. coli B strain (which expresses high levels of lac repressor) by techniques such as electrotransformation or by using calcium chloride. Protocols for these technologies are described in (1). β-galactosidase is a commonly used gene in molecular biology, and various different methods for the construction of β-galactosidase fusion proteins are described in (24). Using these methods or the method described in (1) referred to above, in the proper reading frame, the T7 gene (these do not contain a stop codon that terminates the fusion protein early but in EST1 and EST2). Can be used to generate β-galactosidase appropriately linked to each amino terminus of The DNA molecule encoding the fusion can then be ligated in turn to pKK177-3 plasmid DNA under the control of the tac promoter.

  There are at least four different methods that can be used to generate T7 deletion mutants. These methods include the use of specific restriction enzymes where appropriate, creating unique restriction sites by oligonucleotide-mediated mutagenesis, deletion by oligonucleotide-mediated “loop-out” mutagenesis, and strains. Including the occurrence of genetic deletions. These techniques are outlined in (1). The T7 viral genome is obtained as previously described in (22) and (23). Next, the T7 deletion mutant genome is appropriately packed into virus particles in vitro according to the protocol of (28).

  The population of new molecules is based on several known proteases. This allows several different starting points of evolution from which the desired new molecule can arise. The proteases used are HIV protease, polio 3C protease, and subtilisin BPN ′. These protease sequences are introduced into E. coli and expressed as functional enzymes or enzymes (29) and (30) that may have functionality.

  Each protease gene is subjected to site-directed mutagenesis as well as in vitro chemical mutagenesis to generate large and heterogeneous putative novel molecular populations. A method for site-directed mutagenesis and in vitro chemical mutagenesis is shown in (1).

  The new mutagenized molecule is then converted into a promoter using the methods described above, eg, bacteriophage lambda PR promoter or PL promoter (for use in the host, these are lambdas that down regulate the production of the new molecule. Repressor is not expressed) or connected to T7 promoter. The promoter / novel molecule population is then linked to a T7A deletion mutant population grown in ET1 host cells according to the procedure described above. Once this operation is complete, a heterogeneous T7A deletion mutant population (T7A / novel molecule) is produced, which is highly heterogeneous due to the fact that it encodes a heterogeneous mutagenized pool of novel molecules. is there.

  The T7A / new molecular population is then propagated on ET1 host cells and transferred to EST1 host cells. Keep the EST1 host cells of the T7A / new molecular population in quiescent non-replicating growth phase Only T7A / novel molecular virus particles that can produce new molecules that restore gp1, gp4 and gp5 function by cleaving the β-galactosidase group from the selected molecule can grow in these hosts. T7A / novel molecular virus particles are added in a continuous manner using a cellstat as described in (31), or repeated aliquots of T7A / novel molecular virus particles are added to the EST1 host in a semi-continuous batch method . Once viral replication occurs, the resulting viral particles are recovered and their DNA is isolated / purified as described in (22) and (23). Isolation of T7 / fragment of novel molecular gene that can hybridize with DNA encoding wild-type novel molecule by PCR amplification if the recognition sequence of primer is not mutated in a suppressive manner Obtained by purification or by isolation / purification of fragments capable of expressing molecules with the desired function. Next, sequences encoding new molecules from T7 / new molecule virus particles that can be propagated in EST1 are expressed in any of several expression systems (1), and then proteases are more accurately functionally characterized. Can be determined. If further selection is required, T7B is linked to the sequence of the new molecule from the T7 / new molecule virion that can be propagated in EST1. Thus, these operations are performed as described above except that T7B is used instead of T7A, ET2 is used instead of ET1, and EST2 is used instead of EST1.

  This manipulation allows the development of proteases that are generally specific for cleaving the carboxy terminal region of β-galactosidase.

Example III
Example III Method for Selecting One or More Novel Proteases Specific for Influenza Hemagglutinin Epitope Example III is a specific example of a heptapeptide sequence from influenza hemagglutinin (HA) site A (amino acids 140-146) (32). Describes a method for creating endopeptidases that cleave. The method uses a gene fusion comprising the T7 gene (its function is down-modulated when complexed during fusion) and genes from other bacteriophages. The selection procedure is based on a novel molecule (endoprotease) that cleaves the influenza HA heptapeptide recognition sequence, thereby up-modulating the function of the T7 product required for viral replication. This embodiment corresponds to a virus positive selection operation. A simplified schematic of the method can be seen in FIGS. 2A, 2B and 2C.

The heptapeptide recognition sequence includes amino acids 140-146 of influenza HA, AICHI / 2/68. The sequence is Lys-Arg-Gly-Pro-Gly-Ser-Gly. Two strains EST1 ^* and EST2 ^* are generated that encode heptapeptide sequences as recognition linkers used in such a way that the factor X recognition sequence (33) and the V8 recognition sequence system (34) are used. In addition, these strains are selected to be protease deletions. In these systems, a specific protease specific sequence is placed between the two genes to be cleaved. The EST1 ^* and EST2 ^* constructs are based on the EST1 and EST2 described in Example I, but contain the specific influenza HA heptapeptide sequences described above. Fusion and new molecular populations are constructed and expressed as described above. The same selection protocol is then continued.

Thus, the T7 gene is released from these constructs after the creation of new molecules that can cleave influenza HA heptapeptides. Since the heptapeptide recognition sequence is the only common sequence between all of the fusions, new molecular selections are directed to proteases that are specific only for influenza HA heptapeptides. As described in EST1 and EST2 per Example I, the first novel molecules selected for EST1 ^*, then select per EST2 ^*. The resulting new molecule contained in T7B is isolated / purified and characterized. One skilled in the art will appreciate that this technique can be used to develop proteases with specificity for recognition sequences other than those used in this example.

Example IV
Method IV of creating a novel hydroxylase based on antibody binding describes a method of creating an organic molecule, a molecule that can hydroxylate R at a specific site. The method employs the use of antibodies that can specifically bind to R-OH. A simplified schematic of the process steps can be seen in FIGS. 3A and 3B.

  Antibodies are complexed during fusion with the catalytic subunit of aspartate transcarbamoylase bound to their constant regions. By binding their antigens, antibodies undergo conformational changes. The bound antibody is cleaved by an endoprotease (designed using the methods described in any of the present applications) that can recognize the conformation of the bound antibody. The released aspartate transcarbamoylase catalytic subunit forms a multi-subunit complex, thus functionally upmodulating aspartate transcarbamoylase activity, thus providing the cell with a selective advantage. Therefore, one can select molecules that can catalyze the desired hydroxylation.

  In this example, the subject reaction is site-specific hydroxylation of the drug RH to ROH as shown in (35). A small amount of the desired product, R-OH, or an analog of this product is produced by isolation or chemical synthesis, so that a monoclonal antibody specific for hydroxylated form ROH is produced by a protocol such as the protocol of (36). Can be produced. The gene of the antibody having the desired specificity is linked to the gene of the catalytic subunit of aspartate transcarbamoylase in the constant part of the heavy chain gene of the antibody. These constructs are then expressed in their functional conformation in E. coli by extension of the techniques (10), (11), and (12). These E. coli are selected for the requirement for functionally active aspartate transcarbamoylase for effective growth and are selected to be protease deficient.

  The new molecular population is based on the hydroxylase cytochrome p-450. The gene is mutated to create a heterogeneous population of mutant hydroxylases by various techniques previously described. These genes are then inserted into a plasmid under the control of the low fidelity T7 replication machinery. The plasmid is transferred to cells expressing the antibody fusion protein. The resulting cells are grown in a chemostat in pyrimidine restricted medium. The gene for the hydroxylase encoded in the selected cells is isolated / purified, cloned, and the functional properties of the appropriate hydroxylase determined.

  One skilled in the art will appreciate that this technique can be used to develop molecules with various functional properties other than the ability to act like a hydroxylase. In addition, one skilled in the art will appreciate that molecules other than antibodies that undergo conformational changes upon binding can be combined by various mechanisms to provide growth advantages or disadvantages.

Example V
Method for Creating a Novel Endopeptidase Using Positive Selection of Cells Example V describes a method for creating an endopeptidase that specifically cleaves a decapeptide sequence from gp120. A schematic is seen in FIGS. 1A and 1B.

  The method involves the selection of three cellular genes, aspartate transcarbamoylase, glutamine synthetase, and tryptophan synthetase in a select molecule along with various genes from other bacteriophages (these functions are complexed during fusion). Use gene fusions with (if down-modulated). The selection procedure cleaves the gp120 decapeptide recognition sequence, thus up-modulating the function of the three cellular genes, thus giving cells containing endopeptidases with the desired function a growth advantage under selective conditions. Based on a new molecule (endoprotease). This example corresponds to a positive cell selection operation.

  The genes for bacteriophage T4 virus particle coat proteins 24 and 18 are fused to the amino and carboxy terminus of glutamine synthetase, respectively, by the protease peptide recognition sequence from gp120 used in Example I. Similarly, virus particle coat proteins 27 and 20 are fused with the gp120 linker to the amino and carboxy terminus of the tryptophan synthetase α subunit, respectively, and gene F from T7 gene 12 and gene F from phiX174 are βp of tryptophan synthetase with the gp120 linker. It fuses to the amino terminus and carboxy terminus of the subunit, respectively. Finally, proteins from bacteriophage T4 genes 23 and 15 were fused with gp120 linkers to the amino terminus and carboxy terminus, respectively, of the catalytic subunit of aspartate transcarbamoylase, and bacteriophage T4 gene 27 and bacteriophage T7 The protein from gene 16 is fused with the gp120 linker to the amino terminus and carboxy terminus, respectively, of the regulatory subunit of aspartate transcarbamoylase. Aspartate transcarbamoylase catalyzes the production of N-carbamoyl-aspartate from carbamoyl phosphate and aspartate.

  These constructs can be assembled with appropriate promoters and expressed using the ligation techniques described in Example I, and then lack of the aspartate transcarbamoylase gene, glutamine synthetase gene, and tryptophan synthetase gene. It is integrated into the E. coli chromosome of the E. coli strain which is a mutant. In addition, these E. coli are selected for functionally active aspartate transcarbamoylase, glutamine synthetase, and tryptophan synthetase requirements for effective growth, and also for protease deletion.

  These E. coli are then grown in media rich in glutamine, tryptophan and pyrimidine. The genes for the three protease-based novel molecules from Example I are mutated as described above. However, in this example, the resulting heterogeneous population of genes that express the putative novel molecule is a high copy plasmid such as the PUC vectors described in (1) (eg, pUC18, pUC 19, pUC118, and pUC119). In turn, this is under the control of the mutant low-fidelity T7 replication machinery. A plasmid whose replication is under the control of the T7 replication machinery is described in (37).

  In this example, the plasmid not only encodes multiple novel molecular genes per plasmid, but also encodes the gene required for T7 replication (38) by mutant gene 5 DNA polymerase. This can result in a mutation rate difference between the population of DNA encoding the E. coli chromosome construct and a putative new population of molecules encoded on the plasmid. Selection is then performed in glutamine, tryptophan and pyrimidine restricted media in a chemostat as described in (39) and (40). The selection pressure is then selected for different sub-species of the gene encoding the putative novel molecule, which in turn helps to obtain a variant with high activity for the desired reaction (41 ) In a chemostat (eg low levels of glutamine, tryptophan and pyrimidine in the medium for high selective pressure, and high levels of glutamine, tryptophan and pyrimidine in the medium for low selective pressure). Cycle between environment and low selective pressure environment.

  Finally, evolutionary resetting can be used to further stabilize selected molecule constructs over time. Cells are periodically cycled during the incubation, in three different media: pyrimidine-restricted medium containing glutamine and tryptophan (assay for mutations that produce aspartate transcarbamoylase function), glutamine-restricted medium containing pyrimidine and tryptophan (glutamine). Analyze for mutations that produce synthetase function) and their ability to grow in tryptophan-restricted media containing pyrimidine and glutamine (assay for mutations that produce tryptophan synthetase function). Such methods can be used to analyze the occurrence of unusable variants.

At each time point where a selection molecule variant is seen to occur, the plasmid is isolated / purified as described in the previous example, and then transformed into an aliquot of E. coli encoding the first selection molecule. Used to switch. This allows the selected molecular construct to be evolutionarily reset while maintaining the evolutionary progression of the new molecular population.
The reset population can then be reintroduced into the chemostat. If a novel molecule of the desired specificity is expressed, cells having a gene encoding the desired novel molecule are selected in a chemostat and the plasmid carrying the novel molecule is isolated / as described above. It can be purified.

  Other enzymes or pathways that can synthesize compounds synthesized by aspartate transcarbamoylase, glutamine synthetase, and tryptophan synthetase, or that can end the need for that compound, are preferably non-functional, by deletion It should be. For example, a deletion mutant of glutamine-keto acid transaminase should be used. This is because the enzyme catalyzes the synthesis of glutamine from 2-keto-glutaramate. Also, there should be no mechanism for the synthesis of tryptophan from indole pyruvate or serine. The desired host cells for selection will only grow effectively if glutamine, tryptophan and pyrimidine are added to the medium. Following selection, the genes for the endoprotease of interest are isolated / purified, cloned, and their expression products are characterized.

  One skilled in the art will appreciate that this technique can be used to develop proteases that have specificity for recognition sequences other than those used in the examples.

Example VI
Method for Creating a Novel Endopeptidase Using Negative Cell Selection Example VI describes a method for creating an endopeptidase that specifically cleaves a decapeptide sequence from gp120 at a lower turnover rate than wild type HIV protease. This method uses recombinant β-galactosidase with a decapeptide recognition sequence. The selection method is a novel molecule (endo) that cleaves the gp120 decapeptide recognition sequence to down-modulate β-galactosidase activity, resulting in a growth disadvantage under selective conditions for cells containing endopeptidases with the desired HIV protease activity. Peptidase). This example represents a negative cell selection method.

  There are many known single amino acid changes to HIV protease that can enzymatically inactivate the protease [see (5) and (42)]. One of these inactive missense HIV proteases or a mixture thereof is mutated. In this example, an enzymatically active β-galactosidase containing the HIV protease decapeptide recognition sequence is used, which renders the β-galactosidase molecule inactive by cleavage (29).

  According to the protocol of (29), the cell line is grown to produce E. coli strain MC1061 containing a plasmid having a β-galactosidase construct and a mutant inactive HIV protease (eg Asp-29 → Gly). First, using these methods, a strain containing the HIV protease inactive mutant described in (5) or (42) or a combination of two or more thereof is prepared. These cell lines are then grown to isolate / purify the plasmid (1). The plasmid DNA is then subjected to mutagenesis as already described in (1), or the plasmid is transfected into a strain such as E. coli LE30 mutD strain with a high mutation rate and mutagenized (42).

  The mutated plasmid is then transfected into E. coli strain MC1061 with the desired β-galactosidase construct (29) (1). These cells are then selected for negative cell growth as described in (43).

  One skilled in the art will appreciate that this method can also be used to develop proteases with specificity for recognition sequences other than those used in this example.

Example VII
Method for Creating a Novel Endopeptidase Using Positive Cell Selection Example VII describes a method for obtaining an endopeptidase that specifically cleaves a gp120-derived decapeptide sequence at a lower turnover rate than the wild type HIV protease. This method uses a fusion protein of the catalytic subunit of aspartate transcarbamylase. A selection method is a novel method that cleaves the gp120 decapeptide recognition sequence to upmodulate aspartate transcarbamylase activity, thereby conferring a growth advantage under selective conditions on cells containing endopeptidases with the desired HIV protease activity. Based on molecules (endopeptidases). This example represents a positive cell selection method.

  As in the previous examples, from one or a combination of HIV protease mutants with one amino acid change that is enzymatically inactive and previously subjected to mutagenesis (as described above) Get a molecule with a function. However, in this example, the catalytic subunit of aspartate transcarbamylase having β-galactosidase attached to its amino terminus via the HIV protease recognition decapeptide described in (29) and the HIV protease recognition decapeptide are used. Then, a fusion with the gene 10 (capsid protein) derived from bacteriophage T7 attached to the carboxy terminus is created (1).

  The mutated population of HIV missense mutants is a deletion mutant of the aspartate transcarbamylase catalytic subunit and correctly positioned on the host chromosome using the method described in the previous example (promoted). ) Transfect into modified MC1061 cells containing aspartate transcarbamylase fusion. In addition, these host cells are selected using functional aspartate transcarbamylase requirements for effective growth and using protease deletions. Selection for expression of molecules capable of cleaving the fusion and releasing aspartate transcarbamylase enzyme activity is then performed in a chemostat (39). One skilled in the art will appreciate that this method can also be used to develop proteases with specificity for recognition sequences other than those used in this example.

Example VIII
Method for Creation of Novel HIV Proteases Using Negative Cell Selection This example is similar to Example VI. However, in this example, wild type HIV protease is used as a starting point in place of the enzymatically inactive missense mutant. In addition, the HIV decapeptide recognition sequence is changed to one of the following sequences, each of which is used separately for parallel selection.

IN: integrase protein *: N-terminal product (p6 *) from the pol reading frame has no new name, but otherwise the cleavage site in HIV gag-pol polyprotein is named by the new nomenclature (2) Has been. CA ⁺ represents the capsid protein p25 (24) extended at the C-terminus. ◎
☆: relative value of V _max / K _m were determined by competition experiments. Each value is an average of at least 3 tests with a reproducibility of ± 20%.

  The sequences of BI-P-140, BI-P-138, BI-P-144, BI-P-127 and BI-P-102 (sequence set A) are shown to be cleaved by HIV protease at various rates. However, if the cleavage rate can be detected, all are much slower than that of BI-P-136 (44). Subsequently, negative cell selection was performed using the decapeptide recognition sequence derived from sequence set A. Four of the five sequences are the native sequence of HIV protease, but are processed by the enzyme at a rate that is a fraction of the rate of the BI-P-136 substrate decapeptide. Cells containing these four sequences are prepared that are inserted into β-galactosidase [in a manner similar to (29)] and express HIV protease. These cells may be considered “leaky” auxotrophs to varying degrees. β-galactosidase is first cleaved and consequently inactivated at various levels in cells with these constructs. The fifth sequence, a recognition sequence from avian sarcoma-leukemia virus, is not specifically cleaved by HIV protease (44) and (45). In addition, these strains are selected using functional β-galactosidase requirements for efficient growth and protease deletion. Using various concentrations of antibiotics and lactose, HIV protease mutants with increasing turnover rates for various substrate decapeptides are obtained under negative selection conditions.

  With higher concentrations of lactose and antibiotics, more leaky auxotrophs can be grown with lactose and then killed with penicillin, resulting in a tighter negative selection pressure. This selects HIV protease mutants with higher turnover rates for the construct of interest. Mutant proteases from selected cells are then characterized by the in vitro assay described in (29). These cellular clones with the desired specificity are propagated and the plasmid encoding the desired function is isolated / purified as described above.

  One skilled in the art will appreciate that this method can also be used to develop proteases with specificity for recognition sequences other than those used in this example.

Example IX
Method for Creation of Novel HIV Protease Using Positive Cell Selection This example is similar to Example VII. In this example, wild type HIV protease is used as a starting point in place of the enzymatically inactive missense mutant. In addition, the HIV decapeptide recognition sequence is changed to one of the following sequences (44), each of which is used separately for parallel selection.

IN: integrase protein *: N-terminal product (p6 *) from the pol reading frame has no new name, but otherwise the cleavage site in HIV gag-pol polyprotein is named by the new nomenclature (2) Has been. CA ⁺ represents the capsid protein p25 (24) extended at the C-terminus. ◎
☆: relative value of V _max / K _m were determined by competition experiments. Each value is an average of at least 3 tests with a reproducibility of ± 20%.

  The sequences of BI-P-140, BI-P-138, BI-P-144, BI-P-127 and BI-P-102 (sequence set A) are shown to be cleaved by HIV protease at various rates. However, if the cleavage rate can be detected, all are much slower than that of BI-P-136 (44). A positive cell selection (described above) is then performed at various dilution rates and selection pressures in a chemostat, and a protease capable of releasing aspartate transcarbamylase enzyme activity by cleaving the fusion construct at the desired turnover rate. Select cells to express. The mutant HIV protease in the selected cellular clone is then characterized by the in vitro assay described in (29). These cellular clones with the desired specificity are propagated and the plasmid encoding the desired function is isolated / purified as described above.

  One skilled in the art will appreciate that this method can also be used to produce proteases with specificity for recognition sequences other than those used in this example.

Example X
Cell-free selection This example illustrates how each step is performed in a controlled cell-free environment.

  The endopeptidases used are missense mutants of HIV protease such as Asn-25 (5) and (42). The genes for these enzymes are encoded on a plasmid and can be any of the methods described in the previous examples, including chemical mutagenesis, site-directed mutagenesis, and low fidelity replication (eg, growth in MutD strains). Mutate with or.

  The mutagenized enzyme population is produced in E. coli as described above and then isolated / purified (using the method as described in (1)). Isolate / purify the plasmid containing the endopeptidase population (1). Similarly, to express an actin fusion protein linked to β-galactosidase having an HIV protease recognition sequence at its amino terminus (29) and linked to T7 gene 10 via the same HIV protease recognition sequence at its carboxy terminus. Use the protocol. The expressed fusion construct is isolated / purified as described above. Alternatively, the putative novel molecule and actin fusion gene are amplified and expressed as described in (48) using the expression PCR described above. This eliminates all aspects of using live cells.

  The expressed putative novel molecular protein is incubated with the isolated / purified actin fusion protein to cleave the recognition sequence into the novel molecular protease with the desired function. The mixture resulting from the incubation of the putative novel molecule with the actin fusion is then incubated together in the presence of isolated / purified DNase I. DNase is known to be inhibited by 1: 1 complex formation with actin monomers (46, 47). After successful cleavage of the HIV protease recognition sequence, the actin monomer is sterically free to bind to the DNase I enzyme.

  An isolated / purified plasmid (or PCR DNA) encoding a putative new population of molecules is then added to the reaction mixture and incubated. The resulting DNA is isolated / purified from the incubation mixture by the method described in (1). The DNA is then characterized by gel electrophoresis.

  A DNA band corresponding to the undigested plasmid encoding the putative novel molecule is identified and the DNA from this band is isolated / purified (1) and equally divided into 100 samples. Each sample is amplified, a putative new molecule is expressed, and its function is assayed as described above to determine which sample encodes a new molecule capable of inhibiting DNase I. The sample encoding the enzyme activity is further divided until the desired clone is obtained and can be amplified arbitrarily. At this point, the new molecule is characterized by various attributes such as turnover rate.

  If the properties of the new molecule are desired, no further development is necessary. If not desired, the new molecule is further evolved. Using the DNA encoding these novel molecules, the process of mutating the gene population of the novel molecules again, amplifying and retesting the desired function in the expressed protein is repeated. In this way, DNA encoding a novel molecular population undergoes evolutionary progress, and a novel molecule having favorable properties is obtained.

Example XI
Orientation of a novel protease specific for the epitope of influenza hematoglutinin (HA) encoded in the M13 vector using a selective molecule based on aspartate-semialdehyde dehydrogenase (ASD) in E. coli by batch method performed in chemostat The evolved aspartate-semialdehyde dehydrogenase (ASD) catalyzes the production of aspartate 4-semialdehyde from aspartyl 4-phosphate in E. coli. This reaction is NADPH dependent and liberates phosphate groups. The production of aspartate 4-semialdehyde is a branch point from which the four amino acid precursors methionine, threonine, isoleucine, and lysine are generated. Thus, if ASD is non-functional in the cell and there is no alternative pathway to produce either aspartate 4-semialdehyde or any other important metabolite downstream of aspartate 4-semialdehyde If cells are selected, they will not be able to grow unless the 4 amino acids are added to the medium. In addition, the cells are selected to be protease deleted.

  ASD-based selection constructs are made by creating a fusion protein. β-galactosidase is encoded upstream of the ASD sequence and asparagine synthetase is encoded downstream. β-galactosidase and asparagine synthetase are linked to ASD via a decapeptide recognition sequence, Thr-Asp-Gln-Glu-Gln-Thr-Ser-Leu-Tyr-Val, derived from influenza HA site B (32). This fusion protein is encoded on the E. coli chromosome under the control of the strongest promoter that has been shown to confer acceptable background ASD activity. Of the maximum number of fusion proteins available, novel molecules with low turnover but with the correct proteolytic specificity have a selective advantage.

  This method allows growth of cells and replicators in a chemostat, where the coding sequences for the selected molecule and the putative novel molecule are separated, resulting in distinct mutation rates. . Selection is by double selection of symbiotic cells and replicators.

  The starting point for the evolution of the new molecule is that Achromobacter iophagus and Clostridium cleaving predominantly with proteases that recognize specific sequences and are not harmful to the system, such as X-gly and pro-X-gly-pro. Collagenase derived from the Histolyticium and encoded in the genome of phages such as M13. Numerous M13 phage encoding novel molecules are subjected to strong mutagens such as ethyl methanesulfonate, nitrosoguanidine and irradiation. Mutagenesis yields a heterogeneous population which is then incubated in a chemostat with E. coli encoding the selected construct in minimal medium without 4 amino acids. Random mutagenesis confers evolutionary temporal dominance towards new molecular populations compared to selected constructs. In addition, the M13 genome replicates faster than the E. coli chromosome, and the number of mutations in the M13 genome also increases compared to the E. coli chromosome. These M13 molecules with maximal activity are obtained based on their selective growth advantage. A gene encoding a new molecule that appears to be optimal is isolated / purified from the population, mutated again, and incubated with an evolutionary reset population of E. coli cells encoding a fusion protein selection construct in a repeatable batch method To do. This makes it possible to maintain an optimal novel molecule while stably maintaining the fusion protein by renewing the population many times.

Example XII
Specific to the recognition sequence derived from HIV gp120 encoded in the M13 vector using a differential construct, overlapping and a heterogeneous initial population, using a selection construct containing enzymes important for the synthesis of chorismate in E. coli The directed evolutionary chorismate of a novel novel protease is a branch point in the biosynthesis of phenylalanine, tyrosine and tryptophan. Its production is catalyzed by the enzymatic action of chorismate synthase on 3-enolpyruvylshikimate 5-phosphate. The production of chorismate is through a linear, unbranched pathway from 3-deoxy-7-phospho-D-arabinoheptulosonic acid. The pathway involving chorismate and 3-deoxy-7-phospho-D-arabinoheptulosonate includes seven compounds, catalyzed by six different enzymes, one of which is chorismate synthase.

  Each of the six enzymes is selected to require chorismate for efficient growth, and in a different protein fusion selection construct encoded on the E. coli chromosome of the strain selected to be protease-deficient. Used in. The proteolytic recognition sequence of interest, amino acids 271 to 295 of the variable region 3 derived from HIV gp120 (N-N-T-R-K-S-I-R-I-Q-R-G-P-G -R-A-F-V-T-I-G-K-I-G-N), the coding sequence of each enzyme, β-galactosidase, three phospho-2-3-deoxyheptonate aldolases Sandwiched between suitable bulky groups such as proteins from bacteriophage as described in one of the above and other examples. The fusion protein is encoded on the E. coli chromosome under the control of the strongest promoter known to confer acceptable background activity. It is preferred to use a modulating moiety that is heterologous and has no homology to the gene in the E. coli chromosome. Modulator molecules are selected to effectively downmodulate six enzymes, including chorismate synthase. These redundant selection molecules aim at the development of a protease specific for the recognition sequence from gp120, the only common sequence for all fusions.

  The resulting E. coli cell line encodes all of the six fusion-based selection constructs, thus creating an overlapping system. In this system, the biosynthesis of chorismate is inhibited even if 5 out of 6 protein fusions undergo a non-useful deletion. Furthermore, this duplication system works even if the down-modulation of enzyme function in some proteins is not complete. Since each enzyme catalyzes a reaction in the same pathway, each down-modulation in the fusion has a cumulative effect on chorismate production.

  A putative new molecular population is encoded in the M13 phage. Evolutionary starting points for new molecules include the HIV protease subtilisin BPN, polio 3C protease and collagenase from Achromobacter iophagus and Clostridium histolyticium. A large population of such phage is subjected to mutagenesis, giving evolutionary temporal dominance to a putative new molecular population.

  Differential mutation rates for selected constructs and new molecules are created by using different origins of replication and corresponding replication mechanisms. The selection construct is replicated only by the native E. coli holoenzyme complex, whereas the new molecule is replicated by the T7 DNA replication mechanism.

  In order to create M13 phage such that replication of a putative new population of molecules is directed by the T7 mechanism, a double replication mechanism is introduced. M13 is encoded on a circular single-stranded DNA genome. T7 is a linear double-stranded DNA phage. However, during its replication, M13 is produced in a double stranded form. The normal M13 origin is maintained so that the phage can obtain its double-stranded form, and then the T7 replication system is targeted to replicate the gene on the M13 genome.

To do this, the T7 gene required for replication, including mutational low fidelity T7 DNA polymerase, is expressed from either the M13 genome, bacterial chromosome, or plasmid. These genes include low fidelity modified T7 DNA polymerase (gene 5), T7 RNA polymerase and T7 gyrase (gene 4) (38). The T7 replication mechanism is under the control of a strong promoter so that a high copy number is obtained. Since the M13 genome contains a large number of T7 origins of replication, once M13 is double-stranded, multiple copies of the T7 replication machinery are present in the E. coli holoenzyme (very few copies in the cell) for replication of the M13 genome. ) Competes well. The putative novel molecule (s) are located near the T7 replication origin, if bacterial holoenzyme and T7
When both DNA polymerases actively replicate the same M13 genome, the likelihood of low fidelity modified T7 polymerase replicating new molecules increases.

  By increasing the copy number of a putative novel molecular sequence on the M13 genome, the differential mutation rate is also enhanced. These sequences may be complete or incomplete. For example, multiple copies of a complete putative novel molecule will lead to advantageous homologous recombination. In addition, short DNA, particularly DNA encoding important portions of novel molecules such as catalytic sites, may be randomly introduced into the M13 genome, increasing the number of small recombination around the important portion. These small sequences also result in random flanking sequences in several fractions of recombination (since they are randomly distributed throughout the genome).

  E. coli containing the heterogeneous starting population of M13 described above encoding the overlapping system and the novel molecule is grown in a chemostat. The selection pressure is adjusted by adjusting the chemostat environment, particularly the concentrations and flow rates of the amino acids phenylalanine, tyrosine and tryptophan, and is kept constant or circulated periodically. Samples of M13 bacteriophage are periodically removed and assayed for ability to produce new molecules with the desired proteolytic activity.

Example XIII
Evolution Example XIII oriented novel kinase for NR _I from E. coli, the method of the NR _I from E. coli were phosphorylated, causing thereby evolve directing a kinase that can modulate the function of NR _I Describe (2). The evolutionary starting point is encoded on a plasmid that is replicated by a low fidelity T7 replication mechanism. NR _I Phosphorylated allow cell growth in the selection system, so this is an example of a positive cell selection.

An E. coli strain selected from the requirement for glutamine synthetase for efficient growth is modified and constructed as follows. In this strain the glnA-glnL-glnG operon is substantially altered. except a promoter that is recognized by the sigma ^70. The gene for DNaA is replaced with the exception of the glnL and glnG genes. Except for all other copies of the DNaA gene. NR GlnG gene encoding _I is placed under the control of a promoter that is not recognized by the sigma ⁵⁴ recognized by factors such as sigma ^70. A gene (38) required for T7 replication with low fidelity T7 DNA polymerase is inserted, correctly promoted and expressed. Plasmids are constructed and introduced into strains that are replicated by a low fidelity T7 replication mechanism. Thus, the plasmid based on pUC118 is a deletion of the origin of replication that is functionally recognized by the cellular replication machinery and instead contains the major origin of T7 replication, including the promoters φ1.1A and φ1.1B. A heterogeneous set of evolutionary starting points based on bacterial kinases, CheA, SpoIIJ, FrzE, DctB, and AprZ are mutated, inserted into a plasmid, and expressed correctly.

The cells are then incubated in glutamine and nitrogen limited media. Select cells that can grow. In this embodiment, selection occurs through the cascade. Correctly phosphorylated NR _I can act as an enhancer of the recognized promoter glnApZ by sigma ⁵⁴ by binding the upstream DNA sequence. The enhanced glnApZ promoter then allows high level expression of glnA (glutamine synthetase) and DNaA. These proteins then activate a cascade that allows cell growth and replication.

  After selection, the gene for the desired kinase is isolated / purified, cloned, and the expression product is characterized.

One skilled in the art will appreciate that the method can be used to develop a kinase specific for a recognition sequence other than the recognition sequence of this example.

FIG. 2 shows an embodiment of the host selection method of the present invention for the creation of a gene encoding a novel molecule or the directed evolution of the gene. FIG. 1B is a continuation of FIG. 1A showing an embodiment of the host selection method of the present invention for the creation of a gene encoding a novel molecule or the directed evolution of the gene. FIG. 2 shows an embodiment of the viral replicator of the present invention for the creation of a gene encoding a novel molecule or the directed evolution of the gene. FIG. 2 shows an embodiment of the viral replicator of the present invention for the creation of a gene encoding a novel molecule or the directed evolution of the gene. FIG. 2 shows an embodiment of the viral replicator of the present invention for the creation of a gene encoding a novel molecule or the directed evolution of the gene. FIG. 2 shows a further embodiment of the invention for the creation of a novel hydroxylase based on binding interactions or the directed evolution of the hydroxylase. FIG. 2 shows a further embodiment of the invention for the creation of a novel hydroxylase based on binding interactions or the directed evolution of the hydroxylase. It is the figure which showed the cell-free embodiment of this invention. It is the figure which showed the cell-free embodiment of this invention.

Claims (6)

A method for selecting a molecule that catalyzes the cleavage of a peptide, comprising:
(A) in a population of host cells,
(I) comprising a putative molecule or a number of different putative molecules, and (ii) a recognition sequence that binds to the resulting peptide formed by cleavage of the starting peptide by a molecule evolved from the putative molecule A selection molecule (wherein the binding of the generated peptide by the recognition sequence of the selection molecule increases the activity of the growth factor, thereby increasing the proliferation of the cell and / or replicator),
To express
(B) at least one replicator and / or cells in the population undergo at least one division and / or replication cycle and the multiple putative molecules evolve into the molecules that catalyze peptide cleavage As such, the mutagenesis and selection conditions are imposed on the population of host cells to select a replicator or host, provided that the evolved molecule that catalyzes cleavage of the peptide converts the starting peptide into a product peptide Wherein the binding involves binding of the selected molecule to the generated peptide by a recognition sequence, the binding increasing growth factor activity and increasing cell and / or replicator proliferation, and (c) a cell And / or monitoring increased proliferation of the replicator to identify host cells expressing the molecule that catalyze peptide cleavage,
The above method comprising each step.   The method of claim 1, wherein mutagenesis and selection conditions are imposed sequentially on the host cell population.   The method of claim 1, wherein the putative molecule is homogeneous and / or the putative molecule lacks catalytic activity.   The method of claim 1, wherein the molecule that catalyzes the cleavage of a peptide has a higher substrate specificity and / or catalytic activity than a putative molecule.   The method of claim 1, wherein the molecule that catalyzes cleavage of a peptide has a lower substrate specificity and / or catalytic activity than a putative molecule. The method of claim 1, wherein the mutagenesis condition preferentially mutates a nucleic acid encoding a putative molecule.